Methods and compositions for assaying a sample for an aggregant

ABSTRACT

This invention relates to an aggregation sensor useful for the detection and analysis of aggregants in a sample, and methods, articles and compositions relating to such a sensor. The sensor comprises first and second optically active units, where energy may be transferred from an excited state of the first optically active unit to the second optically active unit. The second optically active unit is present in a lesser amount, but its relative concentration is increased upon aggregation, increasing its absorption of energy from the first optically active units. This increase in energy transfer can be detected in variety of formats to produce an aggregation sensing system for various aggregants, including for quantitation. Other variations of the inventions are described further herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/344,942, filed Jan. 31, 2006, now U.S. Pat. No. 7,666,594 issued Feb.23, 2010, which claims the benefit of U.S. Provisional Application No.60/649,024, filed Jan. 31, 2005. The aforementioned applications arehereby incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No.DMR-0097611 awarded by the National Science Foundation and Grant No.N00014-98-0759 awarded by the Office of Naval Research. The U.S.Government has certain rights in this invention.

TECHNICAL FIELD

This invention relates to an aggregation sensor useful for the detectionand analysis of aggregants in a sample, and methods, articles andcompositions relating to such a sensor.

BACKGROUND OF THE INVENTION

Methods for the detection of biomolecules such as nucleic acids arehighly significant not only in identifying specific targets, but also inunderstanding their basic function. Hybridization probe technologies inparticular continue to be one of the most essential elements in thestudy of gene-related biomolecules.^(1,2,3) They are useful for avariety of both commercial and scientific applications, including theidentification of genetic mutations or single nucleotide polymorphisms(SNP's), medical diagnostics, gene delivery, assessment of geneexpression, and drug discovery.^(4,5,6,7)

Conjugated polymers have proven useful as light gathering molecules in avariety of settings. Conjugated polymers soluble in polar media haveproven particularly useful. Water-soluble conjugated polymers such ascationic conjugated polymers (CCPs) have been used in bioassays toimprove detection sensitivity and provide new routes of selectivity inanalyzing biomolecules.

There is a continuing need in the art for methods of detecting andanalyzing particular biomolecules in a sample, and for compositions andarticles of manufacture useful in such methods. There is a need in theart for novel CCPs, for methods of making and using them, and forcompositions and articles of manufacture comprising such compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A). Fluorescence spectra of PFPB in water as a function of[ss-DNA] ([RU]=5×10⁻⁷ M, [ss-DNA]=0 M to 2.7×10⁻⁸ M in 3.0×10⁻⁹ Mincrements, λ_(exc)=380 nm). (B) Normalized photoluminescence spectra ofPFP (a) the absorption (b) and emission (c) spectra of ss-DNA-TR inwater.

FIG. 2. TR emission intensity for PFP/ss-DNA-TR (a) and PFPB/ss-DNA-TR(b) in water (λ_(exc)=380 nm, [ss-DNA-TR]=2.0 E-8 M, TR intensity iscorrected to reflect the difference in optical density for the twopolymers).

FIG. 3. Normalized fluorescence in water of PFPB/PNA-Cy5 (a),PFPB-DNAn/PNA-Cy5 (c) and PFPB-DNAc/PNA-Cy5 (b) ([PNA-Cy5]=2.0 E-8 M,[RU]=1.6 E-7 M, (λ_(exc)=380 nm).

FIG. 4. Block diagram showing a representative example logic device.

FIG. 5. Block diagram showing a representative example of a kit.

FIG. 6. PL spectra of PFPB₇ (1.0×10⁻⁶ M) with 3.6 μL additions of5.1×10⁻⁶ M of 20 by dsDNA (λ_(ex)=380 nm).

FIG. 7. (a) (left) δ for PFPB₁ (♦), PFPB_(2.5) (▴), PFPB₅ (▪) and PFPB₇() as a function of by concentration for 30 by dsDNA. (b) (right) 8 forPFPB₇ () as a function of by concentration for 30 by dsDNA in theconcentration range of 6.0×10⁻¹⁰ M to 3.0×10⁻⁹ M.

FIG. 8. δ as a function of by concentration of 30 by dsDNA for PFPB₇ at[RU]=2.5×10⁻⁷ M (▪), 5.0×10⁻⁷ M (▴), and 1.0×10⁻⁶ M ().

FIG. 9. δ as a function of [b] or [bp] for ssDNA (ss20-Δ; ss30-◯;ss50-⋄; ss75-∇; ss100-□) and dsDNA (ds20-Δ; ds30-◯; ds50-⋄; ds75-∇;ds100-□), respectively.

DETAILED DESCRIPTION OF THE INVENTION

DNA and RNA detection methods are of considerable scientific andtechnological importance.^(8,9,10) Water-soluble conjugated polymers areof particular interest for this purpose because their molecularstructure allows for collective response and, therefore, opticalamplification of fluorescent signals.^(11,12) The large number ofoptically active units along the polymer chain increases the probabilityof light absorption, relative to small molecule counterparts.¹² Facilefluorescence resonance energy transfer (FRET) makes it possible todeliver excitations to fluorophores, which signal the presence of atarget DNA sequence.^(13,14)

Recent studies indicate that energy transfer between segments inconjugated polymers may be substantially more important than along thebackbone.^(15,16) External perturbations that decrease the elongation ofthe backbone, or that bring segments closer together, can therefore beused to substantially modify the emissive properties of a polymer insolution.

Based on these observations, it occurred to us that a small number offluorescent units within a polymer sequence could be activated bystructural changes that compressed or aggregated the polymer chains toultimately change the emission color. We designed a cationic conjugatedpolymer structure that incorporates these design guidelines.Electrostatic complexation with negatively charged DNA can be used toreduce the average intersegment distance. When combined with afluorophore labeled peptide nucleic acid (PNA) strand, the polymer canbe used to design a three color DNA detection assay.

These working examples are demonstrations of a broader principle: thatof an aggregation sensor that provides a signal that varies depending onits state of aggregation. The aggregation sensor comprises firstoptically active units that dominate its spectrum in the absence ofaggregant, and a lesser proportion of second optically active unitscapable of receiving energy from an excited state of the first opticallyactive units. In the presence of an aggregant, the sensor becomesaggregated, and energy is transferred to the second optically activeunits. The emission from the first optically active units decreases. Thesecond optically active units may disperse the energy non-radiatively,may emit light of a characteristic wavelength, or may be used totransfer energy to a subsequent fluorophore, series of fluorophores, orquencher.

The aggregation sensor binds to a class of aggregants through acomponent having an affinity for the aggregant. The affinity componentmay be an ionic region that can interact with an oppositely chargedregion on the aggregant, or the aggregant and aggregation sensor maycomprise members of a binding pair. The examples utilize a polycationicregion in the sensor, and a negatively charged polynucleotide as anaggregant. Presence of the polynucleotide in a sample leads toaggregation of the sensor, causing a change in its emission and energytransfer to second optically active units present in a smaller amount.Those second optically active units may transfer energy to a subsequentoptically active molecule, which is exemplified as a fluorophore, butcan be a quencher.

The inventions described herein are useful for any assay in which asample can be interrogated regarding an aggregant. Typical assaysinvolve determining the presence of the aggregant in the sample or itsrelative amount, or the assays may be quantitative or semi-quantitative.

The methods can be performed on a substrate. The assay can be performedin an array format on a substrate, which can be a sensor. Thesesubstrates may be surfaces of glass, silicon, paper, plastic, or thesurfaces of optoelectronic semiconductors (such as, but not confined to,indium-doped gallium nitride or polyanilines, etc.) employed asoptoelectronic transducers.

The methods of the invention can be performed in multiplex formats. Insome embodiments, a plurality of different probes can be used to detectcorresponding different species of aggregants in a sample through theuse of different signaling chromophores conjugated to the respectiveprobes or through the use of localization of particular probes todeterminable regions of the substrate. Multiplex methods are providedemploying 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 200, 400 or moredifferent probes which can be used simultaneously to assay forcorresponding different aggregants of interest. It is further envisionedthat in certain embodiments, for example in high density arrays, thenumber of different probes used simultaneously could be 500, 1×10³,1×10⁴, 1×10⁵, 1×10⁶ or more different probes.

Before the present invention is described in further detail, it is to beunderstood that this invention is not limited to the particularmethodology, devices, solutions or apparatuses described, as suchmethods, devices, solutions or apparatuses can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention.

Use of the singular forms “a,” “an,” and “the” include plural referencesunless the context clearly dictates otherwise. Thus, for example,reference to “an aggregation sensor” includes a plurality of aggregationsensors, reference to “a probe” includes a plurality of probes, and thelike. Additionally, use of specific plural references, such as “two,”“three,” etc., read on larger numbers of the same subject less thecontext clearly dictates otherwise.

Terms such as “connected,” “attached,” “conjugated” and “linked” areused interchangeably herein and encompass direct as well as indirectconnection, attachment, linkage or conjugation unless the contextclearly dictates otherwise; in one example, the phrase “conjugatedpolymer” is used in accordance with its ordinary meaning in the art andrefers to a polymer containing an extended series of unsaturated bonds,and that context dictates that the term “conjugated” should beinterpreted as something more than simply a direct or indirectconnection, attachment or linkage.

Where a range of values is recited, it is to be understood that eachintervening integer value, and each fraction thereof, between therecited upper and lower limits of that range is also specificallydisclosed, along with each subrange between such values. The upper andlower limits of any range can independently be included in or excludedfrom the range, and each range where either, neither or both limits areincluded is also encompassed within the invention. Where a value beingdiscussed has inherent limits, for example where a component can bepresent at a concentration of from 0 to 100%, or where the pH of anaqueous solution can range from 1 to 14, those inherent limits arespecifically disclosed. Where a value is explicitly recited, it is to beunderstood that values which are about the same quantity or amount asthe recited value are also within the scope of the invention, as areranges based thereon. Where a combination is disclosed, eachsubcombination of the elements of that combination is also specificallydisclosed and is within the scope of the invention. Conversely, wheredifferent elements or groups of elements are disclosed, combinationsthereof are also disclosed. Where any element of an invention isdisclosed as having a plurality of alternatives, examples of thatinvention in which each alternative is excluded singly or in anycombination with the other alternatives are also hereby disclosed; morethan one element of an invention can have such exclusions, and allcombinations of elements having such exclusions are hereby disclosed.

Unless defined otherwise or the context clearly dictates otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the invention, the preferred methods and materials are nowdescribed.

All publications mentioned herein are hereby incorporated by referencefor the purpose of disclosing and describing the particular materialsand methodologies for which the reference was cited. The publicationsdiscussed herein are provided solely for their disclosure prior to thefiling date of the present application. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The term “aggregation” and the like refer to a relative increase in theconcentration of the second optically active subunit(s) of anaggregation sensor within a particular volume, which may be a localizedregion of a larger volume. The term encompasses any form ofaccumulation, compaction, condensing, etc., that increases the abilityto transfer energy from an excited first optically active unit(s) to asecond optically active unit, including without limitation alteration(s)of the conformation of a single aggregation sensor, the bringingtogether of different aggregation sensors, or both.

“Alkyl” refers to a branched, unbranched or cyclic saturated hydrocarbongroup of 1 to 24 carbon atoms optionally substituted at one or morepositions, and includes polycyclic compounds. Examples of alkyl groupsinclude optionally substituted methyl, ethyl, n-propyl, isopropyl,n-butyl, s-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl,n-heptyl, n-octyl, n-decyl, hexyloctyl, tetradecyl, hexadecyl, eicosyl,tetracosyl and the like, as well as cycloalkyl groups such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, adamantyl, and norbornyl. The term “lower alkyl” refers toan alkyl group of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.Exemplary substituents on substituted alkyl groups include hydroxyl,cyano, alkoxy, ═O, ═S, —NO₂, halogen, haloalkyl, heteroalkyl,carboxyalkyl, amine, amide, thioether and —SH.

“Alkoxy” refers to an “-Oalkyl” group, where alkyl is as defined above.A “lower alkoxy” group intends an alkoxy group containing one to six,more preferably one to four, carbon atoms.

“Alkenyl” refers to a branched, unbranched or cyclic hydrocarbon groupof 2 to 24 carbon atoms containing at least one carbon-carbon doublebond optionally substituted at one or more positions. Examples ofalkenyl groups include ethenyl, 1-propenyl, 2-propenyl (allyl),1-methylvinyl, cyclopropenyl, 1-butenyl, 2-butenyl, isobutenyl,1,4-butadienyl, cyclobutenyl, 1-methylbut-2-enyl, 2-methylbut-2-en-4-yl,prenyl, pent-1-enyl, pent-3-enyl, 1,1-dimethylallyl, cyclopentenyl,hex-2-enyl, 1-methyl-1-ethylallyl, cyclohexenyl, heptenyl,cycloheptenyl, octenyl, cyclooctenyl, decenyl, tetradecenyl,hexadecenyl, eicosenyl, tetracosenyl and the like. Preferred alkenylgroups herein contain 2 to 12 carbon atoms. The term “lower alkenyl”intends an alkenyl group of 2 to 6 carbon atoms, preferably 2 to 4carbon atoms. The term “cycloalkenyl” intends a cyclic alkenyl group of3 to 8, preferably 5 or 6, carbon atoms. Exemplary substituents onsubstituted alkenyl groups include hydroxyl, cyano, alkoxy, ═O, ═S,—NO₂, halogen, haloalkyl, heteroalkyl, amine, thioether and —SH.

“Alkenyloxy” refers to an “-Oalkenyl” group, wherein alkenyl is asdefined above.

“Alkylaryl” refers to an alkyl group that is covalently joined to anaryl group. Preferably, the alkyl is a lower alkyl. Exemplary alkylarylgroups include benzyl, phenethyl, phenopropyl, 1-benzylethyl,phenobutyl, 2-benzylpropyl and the like.

“Alkylaryloxy” refers to an “-Oalkylaryl” group, where alkylaryl is asdefined above.

“Alkynyl” refers to a branched or unbranched hydrocarbon group of 2 to24 carbon atoms containing at least one —C≡C— triple bond, optionallysubstituted at one or more positions. Examples of alkynyl groups includeethynyl, n-propynyl, isopropynyl, propargyl, but-2-ynyl,3-methylbut-1-ynyl, octynyl, decynyl and the like. Preferred alkynylgroups herein contain 2 to 12 carbon atoms. The term “lower alkynyl”intends an alkynyl group of 2 to 6, preferably 2 to 4, carbon atoms, andone —C≡C— triple bond. Exemplary substituents on substituted alkynylgroups include hydroxyl, cyano, alkoxy, ═O, ═S, —NO₂, halogen,haloalkyl, heteroalkyl, amine, thioether and —SH.

“Amide” refers to —C(O)NR′R″, where R′ and R″ are independently selectedfrom hydrogen, alkyl, aryl, and alkylaryl.

“Amine” refers to an —N(R′)R″ group, where R′ and R″ are independentlyselected from hydrogen, alkyl, aryl, and alkylaryl.

“Aryl” refers to an aromatic group that has at least one ring having aconjugated pi electron system and includes carbocyclic, heterocyclic,bridged and/or polycyclic aryl groups, and can be optionally substitutedat one or more positions. Typical aryl groups contain 1 to 5 aromaticrings, which may be fused and/or linked. Exemplary aryl groups includephenyl, furanyl, azolyl, thiofuranyl, pyridyl, pyrimidyl, pyrazinyl,triazinyl, biphenyl, indenyl, benzofuranyl, indolyl, naphthyl,quinolinyl, isoquinolinyl, quinazolinyl, pyridopyridinyl,pyrrolopyridinyl, purinyl, tetralinyl and the like. Exemplarysubstituents on optionally substituted aryl groups include alkyl,alkoxy, alkylcarboxy, alkenyl, alkenyloxy, alkenylcarboxy, aryl,aryloxy, alkylaryl, alkylaryloxy, fused saturated or unsaturatedoptionally substituted rings, halogen, haloalkyl, heteroalkyl, —S(O)R,sulfonyl, —SO₃R, —SR, —NO₂, —NRR′, —OH, —CN, —C(O)R, —OC(O)R, —NHC(O)R,—(CH₂)_(n)CO₂R or —(CH₂)_(n)CONRR′ where n is 0-4, and wherein R and R′are independently H, alkyl, aryl or alkylaryl.

“Aryloxy” refers to an “-Oaryl” group, where aryl is as defined above.

“Carbocyclic” refers to an optionally substituted compound containing atleast one ring and wherein all ring atoms are carbon, and can besaturated or unsaturated.

“Carbocyclic aryl” refers to an optionally substituted aryl groupwherein the ring atoms are carbon.

“Halo” or “halogen” refers to fluoro, chloro, bromo or iodo. “Halide”refers to the anionic form of the halogens.

“Haloalkyl” refers to an alkyl group substituted at one or morepositions with a halogen, and includes alkyl groups substituted withonly one type of halogen atom as well as alkyl groups substituted with amixture of different types of halogen atoms. Exemplary haloalkyl groupsinclude trihalomethyl groups, for example trifluoromethyl.

“Heteroalkyl” refers to an alkyl group wherein one or more carbon atomsand associated hydrogen atom(s) are replaced by an optionallysubstituted heteroatom, and includes alkyl groups substituted with onlyone type of heteroatom as well as alkyl groups substituted with amixture of different types of heteroatoms. Heteroatoms include oxygen,sulfur, and nitrogen. As used herein, nitrogen heteroatoms and sulfurheteroatoms include any oxidized form of nitrogen and sulfur, and anyform of nitrogen having four covalent bonds including protonated forms.An optionally substituted heteroatom refers to replacement of one ormore hydrogens attached to a nitrogen atom with alkyl, aryl, alkylarylor hydroxyl.

“Heterocyclic” refers to a compound containing at least one saturated orunsaturated ring having at least one heteroatom and optionallysubstituted at one or more positions. Typical heterocyclic groupscontain 1 to 5 rings, which may be fused and/or linked, where the ringseach contain five or six atoms. Examples of heterocyclic groups includepiperidinyl, morpholinyl and pyrrolidinyl. Exemplary substituents foroptionally substituted heterocyclic groups are as for alkyl and aryl atring carbons and as for heteroalkyl at heteroatoms.

“Heterocyclic aryl” refers to an aryl group having at least 1 heteroatomin at least one aromatic ring. Exemplary heterocyclic aryl groupsinclude furanyl, thienyl, pyridyl, pyridazinyl, pyrrolyl, N-loweralkyl-pyrrolo, pyrimidyl, pyrazinyl, triazinyl, tetrazinyl, triazolyl,tetrazolyl, imidazolyl, bipyridyl, tripyridyl, tetrapyridyl, phenazinyl,phenanthrolinyl, purinyl and the like.

“Hydrocarbyl” refers to hydrocarbyl substituents containing 1 to about20 carbon atoms, including branched, unbranched and cyclic species aswell as saturated and unsaturated species, for example alkyl groups,alkylidenyl groups, alkenyl groups, alkylaryl groups, aryl groups, andthe like. The term “lower hydrocarbyl” intends a hydrocarbyl group ofone to six carbon atoms, preferably one to four carbon atoms.

A “substituent” refers to a group that replaces one or more hydrogensattached to a carbon or nitrogen. Exemplary substituents include alkyl,alkylidenyl, alkylcarboxy, alkoxy, alkenyl, alkenylcarboxy, alkenyloxy,aryl, aryloxy, alkylaryl, alkylaryloxy, —OH, amide, carboxamide,carboxy, sulfonyl, ═O, ═S, —NO₂, halogen, haloalkyl, fused saturated orunsaturated optionally substituted rings, —S(O)R, —SO₃R, —SR, —NRR′,—OH, —CN, —C(O)R, —OC(O)R, —NHC(O)R, —(CH₂)_(n)CO₂R or —(CH2)_(n)CONRR′where n is 0-4, and wherein R and R′ are independently H, alkyl, aryl oralkylaryl. Substituents also include replacement of a carbon atom andone or more associated hydrogen atoms with an optionally substitutedheteroatom.

“Sulfonyl” refers to —S(O)₂R, where R is alkyl, aryl, —C(CN)═C-aryl,—CH₂CN, alkylaryl, or amine.

“Thioamide” refers to —C(S)NR′R″, where R′ and R″ are independentlyselected from hydrogen, alkyl, aryl, and alkylaryl.

“Thioether” refers to —SR, where R is alkyl, aryl, or alkylaryl.

As used herein, the term “binding pair” refers to first and secondmolecules that bind specifically to each other with greater affinitythan to other components in the sample. The binding between the membersof the binding pair is typically noncovalent. Exemplary binding pairsinclude immunological binding pairs (e.g. any haptenic or antigeniccompound in combination with a corresponding antibody or binding portionor fragment thereof, for example digoxigenin and anti-digoxigenin,fluorescein and anti-fluorescein, dinitrophenol and anti-dinitrophenol,bromodeoxyuridine and anti-bromodeoxyuridine, mouse immunoglobulin andgoat anti-mouse immunoglobulin) and nonimmunological binding pairs(e.g., biotin-avidin, biotin-streptavidin, hormone [e.g., thyroxine andcortisol]-hormone binding protein, receptor-receptor agonist orantagonist (e.g., acetylcholine receptor-acetylcholine or an analogthereof) IgG-protein A, lectin-carbohydrate, enzyme-enzyme cofactor,enzyme-enzyme-inhibitor, and complementary polynucleotide pairs capableof forming nucleic acid duplexes) and the like. One or both member ofthe binding pair can be conjugated to additional molecules.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used interchangeably herein to refer to apolymeric form of nucleotides of any length, and may compriseribonucleotides, deoxyribonucleotides, analogs thereof, or mixturesthereof. These terms refer only to the primary structure of themolecule. Thus, the terms includes triple-, double- and single-strandeddeoxyribonucleic acid (“DNA”), as well as triple-, double- andsingle-stranded ribonucleic acid (“RNA”). It also includes modified, forexample by alkylation, and/or by capping, and unmodified forms of thepolynucleotide.

Whether modified or unmodified, in some embodiments a target nucleotidehas a polyanionic backbone, preferably a sugar-phosphate background, ofsufficient negative charge to electrostatically interact with apolycationic multichromophore in the methods described herein, althoughother forces may additionally participate in the interaction. A sensorpolynucleotide can be used that is a peptide nucleic acid (PNA),although other polynucleotides which minimally interact with themultichromophore in the absence of target can be used in such a format.Alternatively, other embodiments utilize a negatively charged sensorpolynucleotide (for example a DNA or RNA probe). Suitable hybridizationconditions for a given assay format can be determined by one of skill inthe art; nonlimiting parameters which may be adjusted includeconcentrations of assay components, pH, salts used and theirconcentration, ionic strength, temperature, etc.

More particularly, the terms “polynucleotide,” “oligonucleotide,”“nucleic acid” and “nucleic acid molecule” includepolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA,and mRNA, whether spliced or unspliced, any other type of polynucleotidewhich is an N- or C-glycoside of a purine or pyrimidine base, and otherpolymers containing a phosphate or other polyanionic backbone, and othersynthetic sequence-specific nucleic acid polymers providing that thepolymers contain nucleobases in a configuration which allows for basepairing and base stacking, such as is found in DNA and RNA. There is nointended distinction in length between the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and theseterms are used interchangeably herein. These terms refer only to theprimary structure of the molecule. Thus, these terms include, forexample, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′ P5′phosphoramidates, 2′-O-alkyl-substituted RNA, double- andsingle-stranded DNA, as well as double- and single-stranded RNA, andhybrids thereof including for example hybrids between DNA and RNA, andalso include known types of modifications, for example, labels,alkylation, “caps,” substitution of one or more of the nucleotides withan analog, internucleotide modifications such as, for example, thosewith negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (including enzymes (e.g. nucleases), toxins,antibodies, signal peptides, poly-L-lysine, etc.), those withintercalators (e.g., acridine, psoralen, etc.), those containingchelates (of, e.g., metals, radioactive metals, boron, oxidative metals,etc.), those containing alkylators, those with modified linkages (e.g.,alpha anomeric nucleic acids, etc.), as well as unmodified forms of thepolynucleotide or oligonucleotide.

It will be appreciated that, as used herein, the terms “nucleoside” and“nucleotide” will include those moieties which contain not only theknown purine and pyrimidine bases, but also other heterocyclic baseswhich have been modified. Such modifications include methylated purinesor pyrimidines, acylated purines or pyrimidines, or other heterocycles.Modified nucleosides or nucleotides can also include modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen, aliphatic groups, or are functionalized asethers, amines, or the like. The term “nucleotidic unit” is intended toencompass nucleosides and nucleotides.

Furthermore, modifications to nucleotidic units include rearranging,appending, substituting for or otherwise altering functional groups onthe purine or pyrimidine base which form hydrogen bonds to a respectivecomplementary pyrimidine or purine. The resultant modified nucleotidicunit optionally may form a base pair with other such modifiednucleotidic units but not with A, T, C, G or U. Abasic sites may beincorporated which do not prevent the function of the polynucleotide;preferably the polynucleotide does not comprise abasic sites. Some orall of the residues in the polynucleotide can optionally be modified inone or more ways.

Standard A-T and G-C base pairs form under conditions which allow theformation of hydrogen bonds between the N3-H and C4-oxy of thymidine andthe N1 and C6-NH2, respectively, of adenosine and between the C2-oxy, N3and C4-NH2, of cytidine and the C2-NH2, N′—H and C6-oxy, respectively,of guanosine. Thus, for example, guanosine(2-amino-6-oxy-9-β-D-ribofuranosyl-purine) may be modified to formisoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Suchmodification results in a nucleoside base which will no longereffectively form a standard base pair with cytosine. However,modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine)to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine)results in a modified nucleotide which will not effectively base pairwith guanosine but will form a base pair with isoguanosine. Isocytosineis available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine maybe prepared by the method described by Switzer et al. (1993)Biochemistry 32:10489-10496 and references cited therein;2′-deoxy-5-methyl-isocytidine may be prepared by the method of Tor etal. (1993) J. Am. Chem. Soc. 115:4461-4467 and references cited therein;and isoguanine nucleotides may be prepared using the method described bySwitzer et al. (1993), supra, and Mantsch et al. (1993) Biochem.14:5593-5601, or by the method described in U.S. Pat. No. 5,780,610 toCollins et al. Other nonnatural base pairs may be synthesized by themethod described in Piccirilli et al. (1990) Nature 343:33-37 for thesynthesis of 2,6-diaminopyrimidine and its complement(1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione). Other suchmodified nucleotidic units which form unique base pairs are known, suchas those described in Leach et al. (1992) J. Am. Chem. Soc.114:3675-3683 and Switzer et al., supra.

“Complementary” or “substantially complementary” refers to the abilityto hybridize or base pair between nucleotides or nucleic acids, such as,for instance, between a sensor peptide nucleic acid or polynucleotideand a target polynucleotide. Complementary nucleotides are, generally, Aand T (or A and U), or C and G. Two single-stranded polynucleotides orPNAs are said to be substantially complementary when the bases of onestrand, optimally aligned and compared and with appropriate insertionsor deletions, pair with at least about 80% of the bases of the otherstrand, usually at least about 90% to 95%, and more preferably fromabout 98 to 100%.

Alternatively, substantial complementarity exists when a polynucleotideor PNA will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least about 65% complementary over a stretch of at least 14 to 25bases, preferably at least about 75%, more preferably at least about 90%complementary. See, M. Kanehisa Nucleic Acids Res. 12:203 (1984).

“Preferential binding” or “preferential hybridization” refers to theincreased propensity of one polynucleotide or PNA to bind to itscomplement in a sample as compared to a noncomplementary polymer in thesample.

Hybridization conditions will typically include salt concentrations ofless than about 1M, more usually less than about 500 mM and preferablyless than about 200 mM. In the case of hybridization between a peptidenucleic acid and a polynucleotide, the hybridization can be done insolutions containing little or no salt. Hybridization temperatures canbe as low as 5° C., but are typically greater than 22° C., moretypically greater than about 30° C., and preferably in excess of about37° C. Longer fragments may require higher hybridization temperaturesfor specific hybridization. Other factors may affect the stringency ofhybridization, including base composition and length of thecomplementary strands, presence of organic solvents and extent of basemismatching, and the combination of parameters used is more importantthan the absolute measure of any one alone. Other hybridizationconditions which may be controlled include buffer type andconcentration, solution pH, presence and concentration of blockingreagents to decrease background binding such as repeat sequences orblocking protein solutions, detergent type(s) and concentrations,molecules such as polymers which increase the relative concentration ofthe polynucleotides, metal ion(s) and their concentration(s),chelator(s) and their concentrations, and other conditions known in theart.

“Multiplexing” herein refers to an assay or other analytical method inwhich multiple analytes can be assayed simultaneously.

“Having” is an open ended phrase like “comprising” and “including,” andincludes circumstances where additional elements are included andcircumstances where they are not.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not.

The Aggregation Sensor

An aggregation sensor is provided that allows for the detection andanalysis of an aggregant. The aggregation sensor comprises a componentthat can bind to an aggregant or class of aggregants. The interactionmay take place through any means known or discoverable in the art. Theaggregant and aggregation sensor may form members of a binding pair. Theaggregant and aggregation sensor may contain regions of opposite chargethat interact electrostatically. The aggregation sensor comprises alarger number of first optically active units having a first absorptionwavelength at which they can be excited, and can emit light of a firstemission wavelength. The aggregation sensor comprises a lesser number ofsecond optically active units which can absorb light from the excitedstate of the first optically active units. The aggregation sensor cancomprise a ratio of first optically active units to second opticallyactive units of at least three, at least four, at least six, at leastnine, or at least nineteen, or more, so long as a sufficient quantity ofsecond optically active units is provided so that energy may betransferred effectively when the sensor is aggregated.

Aggregation thus leads to an increase in energy transfer from theexcited state formed by a first optically active unit to the secondoptically active unit, thereby decreasing the emission from the firstoptically active unit(s). The second optically active unit maynonradiatively disperse the energy received from the first opticallyactive unit, may emit light of a detectably different wavelength thanthe first optically active unit, or may transfer energy to afluorophore.

Thus aggregation may be assayed by decrease in an emissioncharacteristic of the first optically active unit, by an increase inemission from the second optically active unit(s), or by energy transferto an additional fluorophore that can absorb energy from the secondoptically active unit and emit light or transfer energy to a subsequentfluorophore. A quencher may be substituted for a fluorophore at any stepin the energy transfer scheme after the first optically active unitwhere decrease in emission from the first optically active unit is thedesired assay format.

Aggregation may result from raising the concentration of the aggregationsensor sufficiently to cause the emission from the first opticallyactive unit to decrease completely, due to effectively all of the firstoptically active units in the solution being within energy-transferringdistance of at least one second optically active unit. When anaggregation sensor is used in solution to analyze a sample for anaggregant, it therefore is used at a concentration where the firstoptically active unit still shows a detectable emission and energytransfer to the second optically active unit does not account for allenergy from the excited state of the first optically active unit.

The large number of individual chromophores within the aggregationsensor (and any aggregate formed) can provide amplification of emission;emission from a fluorophore to which energy is transferred can be moreintense when the incident light (the “pump light”) is at a wavelengthwhich is absorbed by the aggregation sensor and transferred to thefluorophore than when the fluorophore is directly excited by the pumplight.

Each type of optically active unit may comprise one or more subunitscapable of absorbing energy. In some embodiments, the optically activeunits form an excited state and emit light of a characteristicwavelength.

In some embodiments, the aggregation sensors used in the presentinvention are polycationic so that they can interact with a biomoleculecomprising multiple anionic groups, e.g. polysaccharides,polynucleotides, peptides, proteins, etc. In some embodiments, theaggregation sensor can interact with a target polynucleotideelectrostatically and thereby bring a signaling chromophore on anuncharged sensor polynucleotide into energy-receiving proximity byvirtue of hybridization between the sensor polynucleotide and the targetpolynucleotide. Any polycationic aggregation sensor that can absorblight and preferably emit or transfer energy can be used in the methodsdescribed. Exemplary aggregation sensors that can be used includeconjugated polymers, saturated polymers or dendrimers incorporatingmultiple chromophores in any viable manner, and semiconductornanocrystals (SCNCs). The conjugated polymers, saturated polymers anddendrimers can be prepared to incorporate multiple cationic species orcan be derivatized to render them polycationic after synthesis;semiconductor nanocrystals can be rendered polycationic by addition ofcationic species to their surface.

In some embodiments, the aggregation sensor is a conjugated polymer(CP). In some embodiments, the CP is one that comprises “lower bandgaprepeat units” of a type and in an amount that contribute a firstabsorption to the polymer in the range of about 450 nm to about 1000 nm.The lower bandgap repeat units may or may not exhibit such an absorptionprior to polymerization, but do introduce that absorption whenincorporated into the conjugated polymer. Exemplary absorption rangesinclude, but are not limited to, wavelengths in the region of 450 nm to500 nm, 500 nm to 550 nm, 550 nm to 600 nm, 600 nm to 700 nm, and 700 nmto 1000 nm. In certain embodiments the polymer forms an excited stateupon contact with incident light having a wavelength including awavelength of about 488 nm, about 532 nm, about 594 nm and about 633 nm.Additionally, useful incident light wavelengths can include, but are notlimited to, 488 nm, 532 nm, 594 nm and 633 nm wavelength light. Suchabsorption characteristics allow the polymer to be excited atwavelengths that produce less background fluorescence in a variety ofsettings, including in analyzing biological samples and imaging and/ordetecting molecules. Shifting the primary absorbance of the CP to alower energy and longer wavelength thus can allow for more sensitive androbust methods in certain formats. Additionally, many commerciallyavailable instruments incorporate imaging components that operate atsuch wavelengths at least in part to avoid such issues. For example,thermal cyclers that perform real-time detection during amplificationreactions and microarray readers are available which operate in thisregion. Providing polymers that absorb in this region allows for theadaptation of detection methods to such formats, and also allowsentirely new methods to be performed. For use in an aggregation sensor,a second optically active species having or contributing an even lowerbandgap absorption is used to receive energy from such a first opticallyactive species, and may be a repeat unit contributing a lower energyabsorption to the polymer.

Incorporation of repeat units that decrease the band gap can produceconjugated polymers with such characteristics. Exemplary optionallysubstituted repeat units which, when incorporated, result in polymersthat absorb light at such wavelengths include 2,1,3-benzothiadiazole,benzoselenadiazole, benzotellurodiazole, naphthoselenadiazole,4,7-di(thien-2-yl)-2,1,3-benzothiadiazole, squaraine dyes, quinoxalines,low bandgap commercial dyes, olefins, and cyano-substituted olefins andisomers thereof. These may be used as the second optically active unitin embodiments of the aggregation sensor.

For example, 2,7-carbazolene-vinylene conjugated polymers have beendescribed with peak absorptions ranging from about 455-485 nm (Morin etal., Chem. Mater. 2004, vol. 16, No. 23, pages 4619-4626). Polymers canbe prepared incorporating benzoselenadiazole with absorption maxima at485 nm. Similarly, polymers incorporating naphthoselenadiazole are knownwith absorption maxima at 550 nm. Polymers incorporating4,7-di(thien-2-yl)-2,1,3-benzothiadiazole are known with absorptionmaxima at about 515 nm. Polymers incorporating cyanovinylenes are knownwith peak absorptions in this region, for example from 372-537 nm, andexhibiting absorption above 700 nm (PFR(1-4)-S, Macromolecules, Vol. 37,No. 14, pages 5265-5273). Preparation of polymers incorporating repeatunits that provide absorption in the spectral region up to 1000 nm hasbeen described (Ajayaghosh, A., et al., Chem. Soc. Rev., 2003, 32, 181;A. Ajayagosh and J. Eldo, Organic Letters, 2001, 3, 2595-2598.) Polymerssoluble in polar media and having absorptions from about 450 nm to about1000 nm can thus be synthesized by incorporation of lower bandgap repeatunits and pendant polar or charged groups as described herein.

In some embodiments, the polymer is one whose absorbance is not shiftedsignificantly in the presence of target or anionic polynucleotide.Desirably, the polymer exhibits no more than about a 15 nm shift in peakabsorbance in the presence of target or anionic polynucleotide; thiscorresponds to no more than about a 0.08 eV shift for a peak absorptionof 480 nm. The polymer may exhibit a peak absorbance shift of about 20,15, 12, 10, 8 or 5 nm or less. The polymer may exhibit a peak absorbanceshift of about 0.10, 0.08, 0.06, 0.04 eV or less. This stability inabsorbance can provide desirable properties in bioassays. Polymers whoseabsorbance shifts excessively depending on assay conditions can lead toundesirable variability.

In some embodiments, a sufficient amount of an appropriate lower bandgaprepeat unit is incorporated into the CP to render it capable ofabsorbing energy at a desired wavelength above 450 nm and providing adetectable signal, where it is desired that the aggregation sensor beexcited in this region.

In some embodiments the polymer can amplify the signal from afluorophore to which it can transfer energy upon excitation. Desirably,the polymer is of a length and comprises a sufficient amount of repeatunits contributing a first absorption wavelength so that upon excitationit transmits sufficient energy to a second or subsequent opticallyactive species (for example another repeat unit contributing a lowerenergy absorption or a fluorophore) so as to achieve a 50% or greaterincrease in light emission from the fluorophore than can be achieved bydirect excitation of the fluorophore in the absence of polymer. Theexact amount of a repeat unit of interest needed to provide the desireddegree of amplification is dependent on a number of factors, and may bedetermined empirically for a given repeat unit. Factors to be consideredinclude the length of the polymer, the molar absorptivity contributed bythe repeat unit, and the interaction between the polymer and theaggregant or biomolecule(s) with which it interacts. The polymer candesirably be of a length and comprise a sufficient amount of a repeatunits of interest to provide a two-fold, three-fold, four-fold,five-fold, or greater increase in emission from an optically activespecies to which it can transfer energy.

The CP can be a copolymer, and may be a block copolymer, a graftcopolymer, or both. A given repeat unit may be incorporated into thepolymer randomly, alternately, periodically and/or in blocks.

The particular size of the polymer is not critical, so long as it isable to absorb light in the relevant region. In some embodiments, thepolymer (which includes oligomers) also desirably is able to transferenergy to a fluorophore. In some embodiments the polymer has a molecularmass of at least about 1,000 Daltons to allow for efficient interactionwith a biomolecule. Typically the polymer will have a molecular mass ofabout 250,000 Daltons or less. An oligomer has at least two repeats of achromophoric unit, and may have a plurality of repeats including 3, 4, 5or more repeats. An oligomer can also comprise a combination of one ormore different chromophoric units, each of which independently may ormay not be repeated.

In some embodiments, the polymer may comprise optionally substitutedfluorenyl repeat units. Polymers comprising fluorenyl repeat unitsexhibiting desirable characteristics and are well studied. However, theabsorption profile of fluorene repeat units shows absorption at shorterwavelengths than is desired in some embodiments described herein. Thusfluorene copolymers additionally incorporating repeat units with lowerbandgaps than fluorene may be desirable in some applications describedherein.

In some embodiments, the polymer may comprise optionally substitutedbiphenylene repeat units. In some embodiments, the polymer may compriseoptionally substituted 2,7-carbazolenevinylene repeat units.

The terms “monomer”, “monomeric unit” and “repeat unit” are usedinterchangeably herein to denote conjugated subunits of a polymer oroligomer. It is to be understood that the repeat units can beincorporated into the polymer at any available position(s) and can besubstituted with one or more different groups. Exemplary substituents onthe repeat units can be selected from alkyl groups, C1-20 alkyl groupsoptionally substituted at one or more positions with S, N, O, P or Siatoms, C4-16 alkyl carbonyloxy, C4-16 aryl(trialkylsiloxy), alkoxy,C1-20 alkoxy, cyano, C1-20 alkylcarbonyloxy, and C1-20 thioether.

The CP contains a sufficient density of solubilizing functionalities torender the overall polymer soluble in a polar medium. Exemplary polarmedia include dimethylsulfoxide (DMSO), dimethylformamide (DMF),ethanol, methanol, isopropanol, dioxane, acetone, acetonitrile,1-methyl-2-pyrrolidinone, formaldehyde, water, and mixtures comprisingthese solvents. The CP is desirably soluble in at least one of thesepolar media, and may be soluble in more than one polar media. The CPpreferably contains at least about 0.01 mol % of the solubilizingfunctionality, and may contain at least about 0.02 mol %, at least about0.05 mol %, at least about 0.1 mol %, at least about 0.2 mol %, at leastabout 0.5 mol %, at least about 1 mol %, at least about 2 mol %, atleast about 5 mol %, at least about 10 mol %, at least about 20 mol %,or at least about 30 mol %. The CP may contain up to 100 mol % of thesolubilizing functionality, and may contain about 99 mol % or less,about 90 mol % or less, about 80 mol % or less, about 70 mol % or less,about 60 mol % or less, about 50 mol % or less, or about 40 mol % orless. Where monomers are polysubstituted, the CP may contain 200 mol %,300 mol %, 400 mol % or more solubilizing functionalities.

The CPs comprise polar groups as solubilizing functionalities linked topolymer subunits to increase polymer solubility in polar media. Any orall of the subunits of the CP may comprise one or more pendantsolubilizing groups. Exemplary polar groups include those introducingone or more dipole moments to the CP, for example halides, hydroxyls,amines, amides, cyano, carboxylic acids, and thiols.

In some embodiments, the polar substituents can be charged groups, forexample cationic or anionic groups. Any suitable cationic or anionicgroups may be incorporated into CPs. When a cationic group isincorporated the polymer is referred to as a cationic conjugated polymer(CCP). Exemplary cationic groups which may be incorporated includeammonium groups, guanidinium groups, histidines, polyamines, pyridiniumgroups, and sulfonium groups. Exemplary anionic groups includecarboxylates, sulfates, and nitrates. The conjugated polymers may have asufficient density of solubilizing polar groups to render them solublein a highly polar solvent such as water and/or methanol. This can beparticularly advantageous for preparing multilayer polymeric devices viasolution processing methods.

The solubilizing functionality may be linked to the conjugated polymerbackbone by a linker, preferably an unconjugated linker, for examplealkyl groups, polyethers, alkylamines, and/or polyamines.

Desirably, the CPs described herein are soluble in aqueous solutions andother polar solvents, and preferably are soluble in water. By“water-soluble” is meant that the material exhibits solubility in apredominantly aqueous solution, which, although comprising more than 50%by volume of water, does not exclude other substances from thatsolution, including without limitation buffers, blocking agents,cosolvents, salts, metal ions and detergents.

One synthetic approach to introducing a charged group into a conjugatedpolymer is as follows. A neutral polymer can be formed by the Suzukicoupling of one or more bis- (or tris-etc.) boronic acid-substitutedmonomers with one or more monomers that have at least two brominesubstitutions on aromatic ring positions. Bromine groups can also beattached to any or all of the monomers via linkers. Polymer ends can becapped by incorporation of a monobrominated aryl group, for examplebromobenzene. Conversion of the polymer to a cationic water-soluble formis accomplished by addition of condensed trimethylamine.

In some embodiments, the CCPs comprise one or more angled linkers with asubstitution pattern (or regiochemistry) capable of perturbing thepolymers' ability to form rigid-rod structures, allowing the CCPs tohave a greater range of three-dimensional structures. The angledlinker(s) are optionally substituted aromatic molecules having at leasttwo separate bonds to other polymer components (e.g., monomers, blockpolymers, end groups) that are capable of forming angles relative to oneanother which disrupt the overall ability of the polymer to form anextended rigid-rod structure (although significant regions exhibitingsuch structure may remain). The angled linkers may be bivalent orpolyvalent.

The angle which the angled linkers are capable of imparting to thepolymeric structure is determined as follows. Where the two bonds toother polymeric components are coplanar, the angle can be determined byextending lines representing those bonds to the point at which theyintersect, and then measuring the angle between them. Where the twobonds to other polymeric components are not coplanar, the angle can bedetermined as follows: a first line is drawn between the two ring atomsto which the bonds attach; two bond lines are drawn, one extending fromeach ring atom in the direction of its respective bond to the otherpolymeric component to which it is joined; the angle between each bondline and the first line is fixed; and the two ring atoms are then mergedinto a single point by shrinking the first line to a zero length; theangle then resulting between the two bond lines is the angle the angledlinker imparts to the CCP.

The angle which an angled linker is capable of imparting to the polymeris typically less than about 155°, and may be less than about 150°, lessthan about 145°, less than about 140°, less than about 135°, less thanabout 130°, less than about 125°, less than about 120°, less than about115°, less than about 110°, less than about 105°, less than about 100°,less than about 95°, less than about 90°, less than about 85°, less thanabout 80°, less than about 75°, less than about 70°, less than about65°, less than about 60°, less than about 55°, or less than about 50°.The angled linker may form an angle to its adjacent polymeric units ofabout 25°, 30°, 35°, 40°, 45°, 50°, 60° or more. The angled linker mayintroduce a torsional twist in the conjugated polymer, thereby furtherdisrupting the ability of the polymer to form a rigid-rod structure. Forangled linkers having an internally rotatable bond, such aspolysubstituted biphenyl, the angled linker must be capable of impartingan angle of less than about 155° in at least one orientation.

For six-membered rings, such angles can be achieved through ortho ormeta linkages to the rest of the polymer. For five-membered rings,adjacent linkages fall within this range. For eight-membered rings,linkages extending from adjacent ring atoms, from alternate ring atoms(separated by one ring atom), and from ring atoms separated by two otherring atoms fall within this range. Ring systems with more than eightring atoms may be used. For polycyclic structures, even more diverselinkage angles can be achieved.

Exemplary linking units which meet these limitations include benzenederivatives incorporated into the polymer in the 1, 2 or 1,3-positions;naphthalene derivatives incorporated into the polymer in the 1,2-, 1,3-,1,6-, 1,7-, 1,8-positions; anthracene derivatives incorporated into thepolymer in the 1,2-, 1,3-, 1,6-, 1,7-, 1,8-, and 1,9-positions; biphenylderivatives incorporated into the polymer in the 2,3-, 2,4-, 2,6-,3,3′-, 3,4-, 3,5-, 2,2′-, 2,3′-, 2,4′-, and 3,4′-positions; andcorresponding heterocycles. The position numbers are given withreference to unsubstituted carbon-based rings, but the same relativepositions of incorporation in the polymer are encompassed in substitutedrings and/or heterocycles should their distribution of substituentschange the ring numbering.

The CCP may contain at least about 0.01 mol % of the angled linker, atleast about 0.02 mol %, at least about 0.05 mol %, at least about 0.1mol %, at least about 0.2 mol %, at least about 0.5 mol %, at leastabout 1 mol %, at least about 2 mol %, at least about 5 mol %, at leastabout 10 mol %, at least about 20 mol %, or at least about 30 mol %. TheCCP may contain about 90 mol % or less, about 80 mol % or less, about 70mol % or less, about 60 mol % or less, about 50 mol % or less, or about40 mol % or less.

An aggregation sensor may be provided in isolated and/or purified form.Any suitable purification or isolation technique may be used, alone orin combination with any other technique. Exemplary techniques includeprecipitation, crystallization, sublimation, chromatography, dialysis,extraction, etc.

A Biomolecule Aggregant

An aggregant to be assayed may be a target biomolecule (e.g., apolysaccharide, a polynucleotide, a peptide, a protein, etc.). In someembodiments the target may interact at least in part electrostaticallywith an aggregation sensor, and may also bind to a sensor biomolecule orprobe in some formats. A target polynucleotide may be a particularspecies of aggregant that is desired to be analyzed, and may bespecifically detected using an aggregation sensor and a sensorpolynucleotide probe, which may be labeled with a fluorophore asdescribed herein. The target polynucleotide can be single-stranded,double-stranded, or higher order, and can be linear or circular.Exemplary single-stranded target polynucleotides include mRNA, rRNA,tRNA, hnRNA, ssRNA or ssDNA viral genomes, although thesepolynucleotides may contain internally complementary sequences andsignificant secondary structure. Exemplary double-stranded targetpolynucleotides include genomic DNA, mitochondrial DNA, chloroplast DNA,dsRNA or dsDNA viral genomes, plasmids, phage, and viroids. The targetpolynucleotide can be prepared synthetically or purified from abiological source. The target polynucleotide may be purified to removeor diminish one or more undesired components of the sample or toconcentrate the target polynucleotide. Conversely, where the targetpolynucleotide is too concentrated for the particular assay, the targetpolynucleotide may be diluted.

Following sample collection and optional nucleic acid extraction, thenucleic acid portion of the sample comprising the target polynucleotidecan be subjected to one or more preparative reactions. These preparativereactions can include in vitro transcription (IVT), labeling,fragmentation, amplification and other reactions. mRNA can first betreated with reverse transcriptase and a primer to create cDNA prior todetection and/or amplification; this can be done in vitro with purifiedmRNA or in situ, e.g. in cells or tissues affixed to a slide. Nucleicacid amplification increases the copy number of sequences of interestsuch as the target polynucleotide. A variety of amplification methodsare suitable for use, including the polymerase chain reaction method(PCR), the ligase chain reaction (LCR), self sustained sequencereplication (3SR), nucleic acid sequence-based amplification (NASBA),the use of Q Beta replicase, reverse transcription, nick translation,and the like.

Where the target polynucleotide is single-stranded, the first cycle ofamplification forms a primer extension product complementary to thetarget polynucleotide. If the target polynucleotide is single-strandedRNA, a polymerase with reverse transcriptase activity is used in thefirst amplification to reverse transcribe the RNA to DNA, and additionalamplification cycles can be performed to copy the primer extensionproducts. The primers for a PCR must, of course, be designed tohybridize to regions in their corresponding template that will producean amplifiable segment; thus, each primer must hybridize so that its 3′nucleotide is paired to a nucleotide in its complementary templatestrand that is located 3′ from the 3′ nucleotide of the primer used toreplicate that complementary template strand in the PCR.

The target polynucleotide can be amplified by contacting one or morestrands of the target polynucleotide with a primer and a polymerasehaving suitable activity to extend the primer and copy the targetpolynucleotide to produce a full-length complementary polynucleotide ora smaller portion thereof. Any enzyme having a polymerase activity thatcan copy the target polynucleotide can be used, including DNApolymerases, RNA polymerases, reverse transcriptases, enzymes havingmore than one type of polymerase activity, and the enzyme can bethermolabile or thermostable. Mixtures of enzymes can also be used.Exemplary enzymes include: DNA polymerases such as DNA Polymerase I(“Pol I”), the Klenow fragment of Pol I, T4, T7, Sequenase® T7,Sequenase® Version 2.0 T7, Tub, Taq, Tth, Pfx, Pfu, Tsp, Tfl, Tli andPyrococcus sp GB-D DNA polymerases; RNA polymerases such as E. coli,SP6, T3 and T7 RNA polymerases; and reverse transcriptases such as AMV,M-MuLV, MMLV, RNAse H⁻ MMLV (SuperScript®), SuperScript® II,ThermoScript®, HIV-1, and RAV2 reverse transcriptases. All of theseenzymes are commercially available. Exemplary polymerases with multiplespecificities include RAV2 and Tli (exo-) polymerases. Exemplarythermostable polymerases include Tub, Taq, Tth, Pfx, Pfu, Tsp, Tfl, Tliand Pyrococcus sp. GB-D DNA polymerases.

Suitable reaction conditions are chosen to permit amplification of thetarget polynucleotide, including pH, buffer, ionic strength, presenceand concentration of one or more salts, presence and concentration ofreactants and cofactors such as nucleotides and magnesium and/or othermetal ions (e.g., manganese), optional cosolvents, temperature, thermalcycling profile for amplification schemes comprising a polymerase chainreaction, and may depend in part on the polymerase being used as well asthe nature of the sample. Cosolvents include formamide (typically atfrom about 2 to about 10%), glycerol (typically at from about 5 to about10%), and DMSO (typically at from about 0.9 to about 10%). Techniquesmay be used in the amplification scheme in order to minimize theproduction of false positives or artifacts produced duringamplification. These include “touchdown” PCR, hot-start techniques, useof nested primers, or designing PCR primers so that they form stem-loopstructures in the event of primer-dimer formation and thus are notamplified. Techniques to accelerate PCR can be used, for examplecentrifugal PCR, which allows for greater convection within the sample,and comprising infrared heating steps for rapid heating and cooling ofthe sample. One or more cycles of amplification can be performed. Anexcess of one primer can be used to produce an excess of one primerextension product during PCR; preferably, the primer extension productproduced in excess is the amplification product to be detected. Aplurality of different primers may be used to amplify different targetpolynucleotides or different regions of a particular targetpolynucleotide within the sample.

Amplified target polynucleotides may be subjected to post amplificationtreatments. For example, in some cases, it may be desirable to fragmentthe target polynucleotide prior to hybridization in order to providesegments which are more readily accessible. Fragmentation of the nucleicacids can be carried out by any method producing fragments of a sizeuseful in the assay being performed; suitable physical, chemical andenzymatic methods are known in the art.

An amplification reaction can be performed under conditions which allowthe sensor polynucleotide to hybridize to the amplification productduring at least part of an amplification cycle. When the assay isperformed in this manner, real-time detection of this hybridizationevent can take place by monitoring for light emission duringamplification.

Real time PCR product analysis (and related real timereverse-transcription PCR) provides a known technique for real time PCRmonitoring that has been used in a variety of contexts, which can beadapted for use with the methods described herein (see, Laurendeau etal. (1999) “TaqMan PCR-based gene dosage assay for predictive testing inindividuals from a cancer family with INK4 locus haploinsufficiency”Clin Chem 45(7):982-6; Laurendeau et al. (1999) “Quantitation of MYCgene expression in sporadic breast tumors with a real-time reversetranscription-PCR assay” Clin Chem 59(12):2759-65; and Kreuzer et al.(1999) “LightCycler technology for the quantitation of bcr/abl fusiontranscripts” Cancer Research 59(13):3171-4).

The Sample

In principle, the sample can be any material suspected of containing anaggregant capable of causing aggregation of the aggregation sensor. Insome embodiments, the sample can be any source of biological materialwhich comprises a biomolecule, for example a polynucleotide, that can beobtained from a living organism directly or indirectly, including cells,tissue or fluid, and the deposits left by that organism, includingviruses, mycoplasma, and fossils. The sample may comprise an aggregantprepared through synthetic means, in whole or in part. Typically, thesample is obtained as or dispersed in a predominantly aqueous medium.Nonlimiting examples of the sample include blood, urine, semen, milk,sputum, mucus, a buccal swab, a vaginal swab, a rectal swab, anaspirate, a needle biopsy, a section of tissue obtained for example bysurgery or autopsy, plasma, serum, spinal fluid, lymph fluid, theexternal secretions of the skin, respiratory, intestinal, andgenitourinary tracts, tears, saliva, tumors, organs, samples of in vitrocell culture constituents (including but not limited to conditionedmedium resulting from the growth of cells in cell culture medium,putatively virally infected cells, recombinant cells, and cellcomponents), and a recombinant library comprising polynucleotidesequences.

The sample can be a positive control sample which is known to containthe aggregant or a surrogate therefor. A negative control sample canalso be used which, although not expected to contain the aggregant, issuspected of containing it (via contamination of one or more of thereagents) or another component capable of producing a false positive,and is tested in order to confirm the lack of contamination by theaggregant of the reagents used in a given assay, as well as to determinewhether a given set of assay conditions produces false positives (apositive signal even in the absence of aggregant in the sample).

The sample can be diluted, dissolved, suspended, extracted or otherwisetreated to solubilize and/or purify an aggregant present or to render itaccessible to reagents. any target polynucleotide which are used in anamplification scheme or to detection reagents. Where the sample containscells, the cells can be lysed or permeabilized to release an aggregantof interest within the cells. One step permeabilization buffers can beused to lyse cells which allow further steps to be performed directlyafter lysis, for example a polymerase chain reaction.

The Probe

One or more probes may be employed that bind to particular species ofaggregants. The probe and the aggregant may form a binding pair thatspecifically bind to each other even with an aggregate. The probe cancomprise a fluorophore or quencher that can participate in energytransfer schemes useful for methods as described herein.

A sensor biomolecule can be used as a probe that can bind to a targetbiomolecule. Exemplary biomolecules include polysaccharides,polynucleotides, peptides, proteins, etc. The sensor biomolecule can beconjugated to a substrate. The sensor may also interact at least in partelectrostatically with a polycationic multichromophore, which may be aconjugated polymer. The sensor biomolecule, the target biomolecule, andthe aggregation sensor when bound together form a detection complex, aform of the aggregant comprising the sensor and aggregant. In someembodiments, a sensor polynucleotide is provided that is complementaryto a target polynucleotide to be assayed, and which does not interactwith the aggregation sensor in the absence of target to a degree thatprecludes the detection of target using the described techniques.Desirably, in some embodiments, the sensor lacks a polyanionic backboneas found in RNA and DNA.

A sensor polynucleotide can be branched, multimeric or circular, but istypically linear, and can contain nonnatural bases. The sensor may belabeled or unlabeled with a detectable moiety.

In some embodiments, the sensor is desirably unlabelled with a moietythat absorbs energy from the aggregation sensor; particularly, thesensor is unlabelled with a fluorophore or quencher that absorbs energyfrom an excited state of the aggregation sensor.

In some embodiments a sensor polynucleotide is labeled with afluorophore that can absorb energy from the aggregation sensor and beused in a fluorescence transfer method for detection of polyanionicspecies.

The sensor may be a PNA, the molecular structures of which are wellknown. PNAs can be prepared with any desired sequence of bases. Specificsensor PNA structures can be custom-made using commercial sources orchemically synthesized.

Fluorophores

In some embodiments, a fluorophore may be employed, for example toreceive energy transferred from an excited state of an optically activeunit, or to exchange energy with a labeled probe, or in multiple energytransfer schemes. Fluorophores useful in the inventions described hereininclude any substance which can absorb energy of an appropriatewavelength and emit or transfer energy. For multiplexed assays, aplurality of different fluorophores can be used with detectablydifferent emission spectra. Typical fluorophores include fluorescentdyes, semiconductor nanocrystals, lanthanide chelates, and greenfluorescent protein.

Exemplary fluorescent dyes include fluorescein, 6-FAM, rhodamine, TexasRed, tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6G,carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy-Chrome, phycoerythrin,PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350, Alexa Fluor®430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor®568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor®660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY®FL, BODIPY® FL-Br₂, BODIPY® 530/550, BODIPY® 558/568, BODIPY® 564/570,BODIPY® 576/589, BODIPY® 581/591, BODIPY® 630/650, BODIPY® 650/665,BODIPY® R6G, BODIPY® TMR, BODIPY® TR, conjugates thereof, andcombinations thereof. Exemplary lanthanide chelates include europiumchelates, terbium chelates and samarium chelates.

A wide variety of fluorescent semiconductor nanocrystals (“SCNCs”) areknown in the art; methods of producing and utilizing semiconductornanocrystals are described in: PCT Publ. No. WO 99/26299 published May27, 1999, inventors Bawendi et al.; U.S. Pat. No. 5,990,479 issued Nov.23, 1999 to Weiss et al.; and Bruchez et al., Science 281:2013, 1998.Semiconductor nanocrystals can be obtained with very narrow emissionbands with well-defined peak emission wavelengths, allowing for a largenumber of different SCNCs to be used as signaling chromophores in thesame assay, optionally in combination with other non-SCNC types ofsignaling chromophores.

Exemplary polynucleotide-specific dyes include acridine orange, acridinehomodimer, actinomycin D, 7-aminoactinomycin D (7-AAD),9-amino-6-chloro-2-methoxyacridine (ACMA), BOBO™-1 iodide (462/481),BOBO™-3 iodide (570/602), BO-PRO™-1 iodide (462/481), BO-PRO™-3 iodide(575/599), 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI),4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI),4′,6-diamidino-2-phenylindole, dilactate (DAPI, dilactate),dihydroethidium (hydroethidine), dihydroethidium (hydroethidine),dihydroethidium (hydroethidine), ethidium bromide, ethidium diazidechloride, ethidium homodimer-1 (EthD-1), ethidium homodimer-2 (EthD-2),ethidium monoazide bromide (EMA), hexidium iodide, Hoechst 33258,Hoechst 33342, Hoechst 34580, Hoechst 5769121, hydroxystilbamidine,methanesulfonate, JOJO™-1 iodide (529/545), JO-PRO™-1 iodide (530/546),LOLO™-1 iodide (565/579), LO-PRO™-1 iodide (567/580), NeuroTrace™435/455, NeuroTrace™ 500/525, NeuroTrace™ 515/535, NeuroTrace™ 530/615,NeuroTrace™ 640/660, OliGreen, PicoGreen® ssDNA, PicoGreen® dsDNA,POPO™-1 iodide (434/456), POPO™-3 iodide (534/570), PO-PRO™-1 iodide(435/455), PO-PRO™-3 iodide (539/567), propidium iodide, RiboGreen®,SlowFade®, SlowFade® Light, SYBR® Green I, SYBR® Green II, SYBR® Gold,SYBR® 101, SYBR® 102, SYBR® 103, SYBR® DX, TO-PRO®-1, TO-PRO®-3,TO-PRO®-5, TOTO®-1, TOTO®-3, YO-PRO®-1 (oxazole yellow), YO-PRO®-3,YOYO®-1, YOYO®-3, TO, SYTOX® Blue, SYTOX® Green, SYTOX® Orange, SYTO® 9,SYTO® BC, SYTO® 40, SYTO® 41, SYTO® 42, SYTO® 43, SYTO® 44, SYTO® 45,SYTO® Blue, SYTO® 11, SYTO® 12, SYTO® 13, SYTO® 14, SYTO® 15, SYTO® 16,SYTO® 20, SYTO® 21, SYTO® 22, SYTO® 23, SYTO® 24, SYTO® 25, SYTO® Green,SYTO® 80, SYTO® 81, SYTO® 82, SYTO® 83, SYTO® 84, SYTO® 85, SYTO®Orange, SYTO® 17, SYTO® 59, SYTO® 60, SYTO® 61, SYTO® 62, SYTO® 63,SYTO® 64, SYTO® Red, netropsin, distamycin, acridine orange,3,4-benzopyrene, thiazole orange, TOMEHE, daunomycin, acridine,pentyl-TOTAB, and butyl-TOTIN. Asymmetric cyanine dyes may be used asthe polynucleotide-specific dye. Other dyes of interest include thosedescribed by Geierstanger, B. H. and Wemmer, D. E., Annu. Rev. Vioshys.Biomol. Struct. 1995, 24, 463-493, by Larson, C. J. and Verdine, G. L.,Bioorganic Chemistry: Nucleic Acids, Hecht, S. M., Ed., OxfordUniversity Press: New York, 1996; pp 324-346, and by Glumoff, T. andGoldman, A. Nucleic Acids in Chemistry and Biology, 2^(nd) ed.,Blackburn, G. M. and Gait, M. J., Eds., Oxford University Press: Oxford,1996, pp 375-441. The polynucleotide-specific dye may be anintercalating dye, and may be specific for double-strandedpolynucleotides. Other dyes and fluorophores are described atwww.probes.com (Molecular Probes, Inc.).

The term “green fluorescent protein” refers to both native Aequoreagreen fluorescent protein and mutated versions that have been identifiedas exhibiting altered fluorescence characteristics, including alteredexcitation and emission maxima, as well as excitation and emissionspectra of different shapes (Delagrave, S. et al. (1995) Bio/Technology13:151-154; Heim, R. et al. (1994) Proc. Natl. Acad. Sci. USA91:12501-12504; Heim, R. et al. (1995) Nature 373:663-664). Delgrave etal. isolated mutants of cloned Aequorea victoria GFP that hadred-shifted excitation spectra. Bio/Technology 13:151-154 (1995). Heim,R. et al. reported a mutant (Tyr66 to His) having a blue fluorescence(Proc. Natl. Acad. Sci. (1994) USA 91:12501-12504).

The Substrate

In some embodiments, an assay component can be located upon a substrate.The substrate can comprise a wide range of material, either biological,nonbiological, organic, inorganic, or a combination of any of these. Forexample, the substrate may be a polymerized Langmuir Blodgett film,functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon,or any one of a wide variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,cross-linked polystyrene, polyacrylic, polylactic acid, polyglycolicacid, poly(lactide coglycolide), polyanhydrides, poly(methylmethacrylate), poly(ethylene-co-vinyl acetate), polysiloxanes, polymericsilica, latexes, dextran polymers, epoxies, polycarbonates, orcombinations thereof. Conducting polymers and photoconductive materialscan be used.

Substrates can be planar crystalline substrates such as silica basedsubstrates (e.g. glass, quartz, or the like), or crystalline substratesused in, e.g., the semiconductor and microprocessor industries, such assilicon, gallium arsenide, indium doped GaN and the like, and includessemiconductor nanocrystals.

The substrate can take the form of a photodiode, an optoelectronicsensor such as an optoelectronic semiconductor chip or optoelectronicthin-film semiconductor, or a biochip. The location(s) of probe(s) onthe substrate can be addressable; this can be done in highly denseformats, and the location(s) can be microaddressable or nanoaddressable.

Silica aerogels can also be used as substrates, and can be prepared bymethods known in the art. Aerogel substrates may be used as freestanding substrates or as a surface coating for another substratematerial.

The substrate can take any form and typically is a plate, slide, bead,pellet, disk, particle, microparticle, nanoparticle, strand,precipitate, optionally porous gel, sheets, tube, sphere, container,capillary, pad, slice, film, chip, multiwell plate or dish, opticalfiber, etc. The substrate can be any form that is rigid or semi-rigid.The substrate may contain raised or depressed regions on which an assaycomponent is located. The surface of the substrate can be etched usingwell known techniques to provide for desired surface features, forexample trenches, v-grooves, mesa structures, or the like.

Surfaces on the substrate can be composed of the same material as thesubstrate or can be made from a different material, and can be coupledto the substrate by chemical or physical means. Such coupled surfacesmay be composed of any of a wide variety of materials, for example,polymers, plastics, resins, polysaccharides, silica or silica-basedmaterials, carbon, metals, inorganic glasses, membranes, or any of theabove-listed substrate materials. The surface can be opticallytransparent and can have surface Si—OH functionalities, such as thosefound on silica surfaces.

The substrate and/or its optional surface can be chosen to provideappropriate characteristics for the synthetic and/or detection methodsused. The substrate and/or surface can be transparent to allow theexposure of the substrate by light applied from multiple directions. Thesubstrate and/or surface may be provided with reflective “mirror”structures to increase the recovery of light.

The substrate and/or its surface is generally resistant to, or istreated to resist, the conditions to which it is to be exposed in use,and can be optionally treated to remove any resistant material afterexposure to such conditions.

Polynucleotide probes can be fabricated on or attached to the substrateby any suitable method, for example the methods described in U.S. Pat.No. 5,143,854, PCT Publ. No. WO 92/10092, U.S. patent application Ser.No. 07/624,120, filed Dec. 6, 1990 (now abandoned), Fodor et al.,Science, 251: 767-777 (1991), and PCT Publ. No. WO 90/15070). Techniquesfor the synthesis of these arrays using mechanical synthesis strategiesare described in, e.g., PCT Publication No. WO 93/09668 and U.S. Pat.No. 5,384,261.

Still further techniques include bead based techniques such as thosedescribed in PCT Appl. No. PCT/US93/04145 and pin based methods such asthose described in U.S. Pat. No. 5,288,514.

Additional flow channel or spotting methods applicable to attachment ofsensor polynucleotides to the substrate are described in U.S. patentapplication Ser. No. 07/980,523, filed Nov. 20, 1992, and U.S. Pat. No.5,384,261. Reagents are delivered to the substrate by either (1) flowingwithin a channel defined on predefined regions or (2) “spotting” onpredefined regions. A protective coating such as a hydrophilic orhydrophobic coating (depending upon the nature of the solvent) can beused over portions of the substrate to be protected, sometimes incombination with materials that facilitate wetting by the reactantsolution in other regions. In this manner, the flowing solutions arefurther prevented from passing outside of their designated flow paths.

Typical dispensers include a micropipette optionally roboticallycontrolled, an ink-jet printer, a series of tubes, a manifold, an arrayof pipettes, or the like so that various reagents can be delivered tothe reaction regions sequentially or simultaneously.

The substrate or a region thereof may be encoded so that the identity ofthe sensor located in the substrate or region being queried may bedetermined. Any suitable coding scheme can be used, for example opticalcodes, RFID tags, magnetic codes, physical codes, fluorescent codes, andcombinations of codes.

Excitation and Detection

Any instrument that provides a wavelength that can excite theaggregation sensor and is shorter than the emission wavelength(s) to bedetected can be used for excitation. Commercially available devices canprovide suitable excitation wavelengths as well as suitable detectioncomponents.

Exemplary excitation sources include a broadband UV light source such asa deuterium lamp with an appropriate filter, the output of a white lightsource such as a xenon lamp or a deuterium lamp after passing through amonochromator to extract out the desired wavelengths, a continuous wave(cw) gas laser, a solid state diode laser, or any of the pulsed lasers.Emitted light can be detected through any suitable device or technique;many suitable approaches are known in the art. For example, afluorimeter or spectrophotometer may be used to detect whether the testsample emits light of a wavelength characteristic of the signalingchromophore upon excitation of the multichromophore.

Incident light wavelengths useful for excitation of aggregation sensorsincluding a plurality of lower bandgap repeat units can include 450 nmto 1000 nm wavelength light. Exemplary useful incident light wavelengthsinclude, but are not limited to, wavelengths of at least about 450, 500,550, 600, 700, 800 or 900 nm, and may be less than about 1000, 900, 800,700, 600, 550 or 500 nm. Exemplary useful incident light in the regionof 450 nm to 500 nm, 500 nm to 550 nm, 550 nm to 600 nm, 600 nm to 700nm, and 700 nm to 1000 nm. In certain embodiments the aggregation sensorforms an excited state upon contact with incident light having awavelength including a wavelength of about 488 nm, about 532 nm, about594 nm and/or about 633 nm. Additionally, useful incident lightwavelengths can include, but are not limited to, 488 nm, 532 nm, 594 nmand 633 nm wavelength light.

Compositions of Matter

Also provided are compositions of matter of any of the moleculesdescribed herein in any of various forms. The aggregation sensorsdescribed herein may be provided in purified and/or isolated form. Theaggregation sensors may be provided in crystalline form.

The aggregation sensors may be provided in solution, which may be apredominantly aqueous solution, which may comprise one or more of theadditional solution components described herein, including withoutlimitation additional solvents, buffers, biomolecules, polynucleotides,fluorophores, etc. The aggregation sensor can be present in solution ata concentration at which a first emission from the first opticallyactive units can be detected in the absence of an aggregant. Thesolution may comprise additional components as described herein,including labeled probes such as fluorescently labeled polynucleotides,specific for a species of a class of aggregants for the aggregationsensor.

The aggregation sensors may be provided in the form of a film. Thecompositions of matter may be claimed by any property described herein,including by proposed structure, by method of synthesis, by absorptionand/or emission spectrum, by elemental analysis, by NMR spectra, or byany other property or characteristic.

Methods of Use

The aggregation sensors provided herein may be employed in a variety ofbiological assays. They may be used to detect the presence of anaggregant in a sample, for example a polynucleotide or otherbiomolecule, and may be used to quantitate the aggregant. In someembodiments, the aggregation sensors bind at least in partelectrostatically to a target biomolecule. The target biomolecule may belabeled or unlabeled. One or more assay components (e.g., probe,aggregation sensor, aggregant) may be bound to a substrate.

The novel aggregation sensors may also be used in biological assays inwhich energy is transferred between one or more of the aggregationsensor, a label on a target aggregant, a label on a probe, and/or afluorescent dye specific for a polynucleotide, in all permutations. Theaggregation sensor may interact at least in part electrostatically withthe sensor and/or the target to form additional complexes, and anincrease in energy transfer with the aggregation sensor may occur uponbinding of the sensor and the target. This method may also be performedon a substrate.

Other variations of such methods are described further herein.

Addition of organic solvents in some cases can result in a decrease inbackground emission by inhibiting nonionic interactions between assaycomponents, for example between the aggregation sensor and a sensorpolynucleotide. The added solvent may be a polar organic solvent, andmay be water miscible, for example an alcohol such as methanol, ethanol,propanol or isopropanol. The added solvent may be one that does notadversely affect the ability of the sensor to hybridize to the target inthe solution, for example 1-methyl-2-pyrrolidinone. The organic solventmay be added in an amount of about 1%, about 2%, about 5%, about 10%, ormore of the total solution, and typically is used within the range ofabout 0.5-10%. Other components may be incorporated into the assaysolution, for example one or more buffers suitable for maintaining a pHsatisfactory for the biological molecules and their desired properties(e.g., ability to hybridize).

In one variation a plurality of fluorophores, which may be directly orindirectly attached or recruited to any other of the assay componentsand/or to a substrate, can be used to exchange energy in an energytransfer scheme. In particular applications, this can provide forsignificant additional selectivity. For example, apolynucleotide-specific dye can be used as an optically active unit, andmay be specific for double-stranded sequences. Energy can be transferredto or from an excited aggregation sensor in certain embodiments. Thecascade of energy transfer can, in principle, be extended to use anynumber of optically active units with compatible absorption and emissionprofiles. In one embodiment of this variation, an intercalating dye thatis specific for double-stranded polynucleotides is used. Theintercalating dye provides the added selective requirement that thesensor and target polynucleotides hybridize before it is recruited tothe detection complex. In the presence of target, the duplex is formed,the dye is recruited, and excitation to or from the dye can occur. Incertain embodiments the methods of using intercalating dye(s) caninclude steps wherein the intercalating dye(s) is in a solution.

In one embodiment a single nucleotide polymorphism (SNP) is detected inan aggregant. In another embodiment expression of a gene is detected inan aggregant. In a further embodiment, a measured result of detecting anaggregant can be used to diagnose a disease state of a patient. In yetanother embodiment the detection method of the invention can furtherinclude a method of diagnosing a disease state. In a related embodiment,the method of diagnosing a disease can include reviewing or analyzingdata relating to the presence of an aggregant and providing a conclusionto a patient, a health care provider or a health care manager, theconclusion being based on the review or analysis of data regarding adisease diagnosis. Reviewing or analyzing such data can be facilitatedusing a computer or other digital device and a network as describedherein. It is envisioned that information relating to such data can betransmitted over the network.

In practicing the methods of the present invention, known molecularbiology techniques are optionally utilized. Exemplary molecular biologytechniques are explained in, for example, Ausubel et al. (Eds.) CurrentProtocols in Molecular Biology, Volumes I, II, and III, (1997), Ausubelet al. (Eds.), Short Protocols in Molecular Biology: A Compendium ofMethods from Current Protocols in Molecular Biology, 5^(th) Ed., JohnWiley & Sons, Inc. (2002), Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press (2000),and Innis et al. (Eds.) PCR Protocols: A Guide to Methods andApplications, Elsevier Science & Technology Books (1990).

Any effective detection method can be used in the various methodsdescribed herein, including optical, spectroscopic, electrical,electrochemical, piezoelectrical, magnetic, Raman scattering, surfaceplasmon resonance, radiographic, colorimetric, calorimetric, etc.Preferably the sensor is or can be rendered optically detectable to ahuman and/or a detection device.

The methods described herein may be used with and incorporated into anapparatus. The methods may be used in conjunction with a commerciallyavailable device. Exemplary commercially available systems andinstruments that can be used in conjunction with an invention disclosedherein include: array systems such as the Affymetrix Genechip® system,Agilent GenePix® Microarray Scanner, Genomic Solutions GeneMachine®,Asper Biotech Genorama™ Quattroimager, and the Bio-Rad VersArray®ChipReader; and real time PCR systems such as the Applied Biosystems7900HT Fast Real-Time PCR System, ABI PRISM® 7000 Sequence DetectionSystem, Applied Biosystems 7500 Real-Time PCR System, Applied Biosystems7300 Real-Time PCR System, Applied Biosystems PRISM® 7700, Bio-Rad MyiQSingle-Color Real-Time PCR Detection System, and the Bio-Rad iCycler iQReal-Time PCR Detection System.

Articles of Manufacture

For the embodiments of the aggregation sensor that are conjugatedpolymers (CPs), those polymers can be incorporated into any article ofmanufacture in which conjugated polymers find use. Exemplary articles ofmanufacture into which the CPs can be incorporated includeoptoelectronic or electronic devices, biosensors, diodes, includingphotodiodes and light-emitting diodes (“LEDs”), optoelectronicsemiconductor chips, semiconductor thin-films, and chips, and can beused in array or microarray form. The CPs can be incorporated into apolymeric photoswitch. The CPs can be incorporated into an opticalinterconnect or a transducer to convert a light signal to an electricalimpulse. The CPs can serve as liquid crystal materials. The CPs may beused as electrodes in electrochemical cells, as conductive layers inelectrochromic displays, as field effective transistors, and as Schottkydiodes.

The CPs can be used as lasing materials. Optically pumped laser emissionhas been reported from MEH-PPV in dilute solution in an appropriatesolvent, in direct analogy with conventional dye lasers [D. Moses, Appl.Phys. Lett. 60, 3215 (1992); U.S. Pat. No. 5,237,582]. Semiconductingpolymers in the form of neat undiluted films have been demonstrated asactive luminescent materials in solid state lasers [F. Hide, M. A.Diaz-Garcia, B. J. Schwartz, M. R. Andersson, Q. Pei, and A. J. Heeger,Science 273, 1833 (1996); N. Tessler, G. J. Denton, and R. H. Friend,Nature 382, 695 (1996)]. The use of semiconducting polymers as materialsfor solid state lasers is disclosed in U.S. Pat. No. 5,881,083 issuedMar. 9, 1999 to Diaz-Garcia et al. and titled “Conjugated Polymers asMaterials for Solid State Lasers.” In semiconducting polymers, theemission is at longer wavelengths than the onset of significantabsorption (the Stokes shift) resulting from inter- and intramolecularenergy transfer. Thus there is minimal self-absorption of the emittedradiation [F. Hide et al., Science 273, 1833 (1996)], so self-absorptiondoes not make the materials lossy. Moreover, since the absorption andemission are spectrally separated, pumping the excited state via the πto π* transition does not stimulate emission, and an inverted populationcan be obtained at relatively low pump power.

Light-emitting diodes can be fabricated incorporating one or more layersof CPs, described herein which may serve as conductive layers. Light canbe emitted in various ways, e.g., by using one or more transparent orsemitransparent electrodes, thereby allowing generated light to exitfrom the device.

The mechanism of operation of a polymer LED requires that carrierinjection be optimized and balanced by matching the electrodes to theelectronic structure of the semiconducting polymer. For optimuminjection, the work function of the anode should lie at approximatelythe top of the valence band, E_(v), (the π-band or highest occupiedmolecular orbital, HOMO) and the work function of the cathode should lieat approximately the bottom of the conduction band, E_(c), (the π*-bandor lowest unoccupied molecular orbital, LUMO).

LED embodiments include hole-injecting and electron-injectingelectrodes. A conductive layer made of a high work function material(above 4.5 eV) may be used as the hole-injecting electrode. Exemplaryhigh work function materials include electronegative metals such as goldor silver, and metal-metal oxide mixtures such as indium-tin oxide. Anelectron-injecting electrode can be fabricated from a low work functionmetal or alloy, typically having a work function below 4.3. Exemplarylow work function materials include indium, calcium, barium andmagnesium. The electrodes can be applied by any suitable method; anumber of methods are known to the art (e.g. evaporated, sputtered, orelectron-beam evaporation).

In some embodiments, polymer light-emitting diodes can be fabricatedusing a semiconducting polymer cast from solution in an organic solventas an emissive layer and a water-soluble (or methanol-soluble)conjugated copolymer as an electron-transport layer (ETL) in the deviceconfiguration: ITO (indium tin oxide)/PEDOT (poly(3,4-ethylenedioxythiophene)/emissive polymer/ETL/Ba/Al.

Any form of conducting layer can be used. Thus, judicious choice ofmonomers as described herein can result in polymers with hole-injectingand/or transporting properties, as well as polymers withelectron-injecting and/or transporting properties. The device geometryand deposition order can be selected based on the type of conductivepolymer being used. More than one type of conductive polymer can be usedin the same multilayer device. A multilayer device may include more thanone layer of electron-injecting conjugated polymers, more than one layerof hole-injecting conjugated polymers, or at least one layer of ahole-injecting polymer and at least one layer of an electron-injectingconjugated polymer.

In PLEDs, the device efficiency is reduced by cathode quenching sincethe recombination zone is typically located near the cathode.^([20]) Theaddition of an ETL moves the recombination zone away from the cathodeand thereby eliminates cathode quenching. In addition, the ETL can serveto block the diffusion of metal atoms, such as barium and calcium, andthereby prevents the generation of quenching centers^([20]) during thecathode deposition process.

In some embodiments, the principal criteria when a soluble conjugatedpolymer is used as an electron transport layer (ETL) in polymerlight-emitting diodes (PLEDs) are the following: (1) The lowestunoccupied molecular orbital (LUMO) of the ETL must be at an energyclose to, or even within the π*-band of the emissive semiconductingpolymer (so electrons can be injected); and (2) The solvent used forcasting the electron injection material must not dissolve the underlyingemissive polymer.

FIG. 4 is a block diagram showing a representative example logic devicethrough which reviewing or analyzing data relating to the presentinvention can be achieved. Such data can be in relation to a disease,disorder or condition in a subject. FIG. 4 shows a computer system (ordigital device) 100 that may be understood as a logical apparatus thatcan read instructions from media 111 and/or network port 105, which canoptionally be connected to server 109 having fixed media 112. The systemshown in FIG. 4 includes CPU 101, disk drives 103, optional inputdevices such as keyboard 115 and/or mouse 116 and optional monitor 107.Data communication can be achieved through the indicated communicationmedium to a server 109 at a local or a remote location. Thecommunication medium can include any means of transmitting and/orreceiving data. For example, the communication medium can be a networkconnection, a wireless connection or an internet connection. It isenvisioned that data relating to the present invention can betransmitted over such networks or connections.

In one embodiment, a computer-readable medium includes a medium suitablefor transmission of a result of an analysis of a biological sample. Themedium can include a result regarding a disease condition or state of asubject, wherein such a result is derived using the methods describedherein.

Similarly, the principal criteria for a polymer based hole transportlayer (HTL) for use in polymer light-emitting diodes (PLEDs) is that thehighest occupied molecular orbital (HOMO) of the HTL must be at anenergy close to, or even within the valence band of the emissivesemiconducting polymer.

Solubility considerations can dictate the deposition order of theparticular CPs and solvents used to produce a desired deviceconfiguration. Any number of layers of CPs with different solubilitiesmay be deposited via solution processing by employing these techniques.

The PLEDs comprising CPs described herein can be incorporated in anyavailable display device, including a full color LED display, a cellphone display, a PDA (personal digital assistant), portable combinationdevices performing multiple functions (phone/PDA/camera/etc.), a flatpanel display including a television or a monitor, a computer monitor, alaptop computer, a computer notepad, and an integrated computer-monitorsystems. The PLEDs may be incorporated in active or passive matrices.

Kits

Kits comprising reagents useful for performing described methods arealso provided.

In some embodiments, a kit comprises an aggregation sensor as describedherein and one or more fluorescently labeled probes specific for aspecies of aggregant of interest. In the presence of the specificaggregant in the sample, the sensor is brought into proximity to theaggregation sensor via binding to the aggregant.

The kit may optionally contain one or more of the following: one or morelabels that can be incorporated into an aggregant; one or moreintercalating dyes; one or more sensor biomolecules, one or moresubstrates which may or may not contain an array, etc.

The components of a kit can be retained by a housing. Instructions forusing the kit to perform a described method can be provided with thehousing, and can be provided in any fixed medium. The instructions maybe located inside the housing or outside the housing, and may be printedon the interior or exterior of any surface forming the housing thatrenders the instructions legible. A kit may be in multiplex form fordetection of one or more different target polynucleotides or otherbiomolecules.

As described herein and shown in FIG. 5, in certain embodiments a kit200 can include a housing 202 for housing various components. As shownin FIG. 5, and described herein, in one embodiment a kit 200 includes anaggregation sensor 206 as described herein and optionally one or morefluorescently labeled probe 207, one or more labels 208 that can beincorporated into an aggregant or other assay component, one or moreintercalating dyes 209, one or more sensor biomolecules 210 and one ormore substrates 212. As shown in FIG. 5, and described herein, the kit200 can optionally include instructions 204. Other embodiments of thekit 200 wherein the components include various additional featuresdescribed herein are within the scope of the invention.

EXAMPLES

The following examples are set forth so as to provide those of ordinaryskill in the art with a complete description of how to make and use thepresent invention, and are not intended to limit the scope of what isregarded as the invention. Efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, etc.) but someexperimental error and deviation should be accounted for. Unlessotherwise indicated, parts are parts by weight, temperature is degreecentigrade and pressure is at or near atmospheric, and all materials arecommercially available.

Example 1 Synthesis of a Conjugated Polymeric Aggregation Sensor

The synthetic approach involved initially a Suzuki copolymerization ofpara-phenylenebisboronic acid with a 95:5 mixture of2,7-dibromo-9,9-bis(6′-bromohexyl)fluorene and4,7-dibromo-2,1,3-benzothiadiazole.¹⁷ Elemental analysis of theresulting polymer is consistent with a chemical composition similar tothe monomer feed. Since the GPC determined molecular weight (M_(n)) was˜11,000 amu, one can estimate that there is, on average, one BT moleculeper polymer chain. In a second step, quarternization of the pendantgroups by addition of NMe₃ provides the polycationic water soluble PFPB(see Scheme 1).

General Details. 1H and ¹³C NMR spectra were collected on Varian ASM 200MHz spectrometers. UV-Vis absorption spectra were recorded on a ShimadzuUV-2401 PC diode array spectrometer. Photoluminescence spectra wereobtained using a Spex Fluorolog 2 spectrometer, using 90 degree angledetection for solution samples. Elemental analysis were performed by theUC Santa Barbara elemental analysis center. Reagents were obtained fromAldrich Co., and used as received.

4,7-dibromo-2,1,3-benzothiadiazole. 2,1,3-benzothiadiazole (6.8 g, 50mmol) in 15 mL of 47% HBr solution was heated to reflux while bromine(24 g, 150 mmol) was added dropwise. At the end of the addition, anextra 10 mL of HBr was added, and the mixture was heated under refluxfor an additional three hours. The mixture was filtered while hot, andthe filtrate was washed with water, 5% sodium bicarbonate and water. Thecrude product was collected, dried, and recrystallized fromchloroform-hexane to afford 4,7-dibromo-2,1,3-benzothiadiazole (9.0 g,61.2%) as yellow crystals. 1H NMR (CDCl₃, 200 MHz): d 7.72 ppm.

Poly[9,9-bis(6′-bromohexyl)fluorene-co-1,4-phenylene-co-4,7-(2,1,3-benzothiadiazole)](PFPB precursor). 2,7-Dibromo-9,9-bis(6′-bromohexyl)fluorene (306.8 mg,0.475 mmol), 4,7-dibromo-2,1,3-benzothiadiazole (7.3 mg, 0.025 mmol),1,4-phenyldiboronic acid (82.9 mg, 0.5 mmol), Pd(PPh3)₄ (8 mg) andpotassium carbonate (830 mg, 6 mmol) were placed in a 25 mL round bottomflask. A mixture of water (3 mL) and THF (6 mL) was added to the flaskand the reaction vessel was degassed. The mixture was refluxed at 85° C.for 20 h under nitrogen, and then precipitated into methanol. Thepolymer was filtered and washed with methanol and acetone, and thendried under vacuum for 24 h to afford the neutral PFPB precursor (180mg, 65.9%), as a light yellow fibrous solid. 1H NMR (200 MHz, CDCl₃): d7.8 (m, 5H), 7.7-7.5 (m, 5H), 3.3 (t, 4H), 2.1 (m, 4H), 1.7 (br, 4H),1.3-1.2 (m, 8H), 0.8 (br, 4H). Elemental analysis: Calcd forC15.025H16.3Br0.95N0.05S0.025: C, 65.79; H, 5.98; N, 0.26. Found: C,64.52; H, 5.39; N, 0.39. GPC (THF, polystyrene standard), Mw: 19,500g/mol; Mn: 11,000 g/mol; PDI: 1.95.

Poly(9,9-bis(6′-N,N,N-trimethylammoniumbromide)hexyl)fluorene-co-1,4-phenylene-co-4,7-(2,1,3-benzothiadiazole)](PFPB). Condensed trimethylamine (2 mL) was added dropwise to a solutionof the neutral precursor polymer (50 mg) in THF (10 mL) at −78° C. Themixture was allowed to warm up to room temperature. The precipitate wasre-dissolved by addition of water (10 mL). After the mixture was cooleddown to −78° C., extra trimethylamine (2 mL) was added and the mixturewas stirred for 24 h at room temperature. After removing most of thesolvent, acetone was added to precipitate PFPB (55 mg, 91.2%) as a lightyellow powder. 1H NMR (200 MHz, CD3OD): d 8.1-7.7 (m, 10H), 3.3-3.2 (t,4H), 3.1 (s, 18H), 2.3 (br, 4H), 1.6 (br, 4H), 1.3 (br, 8H), 0.8 (br,4H).

Example 2 Demonstration of an Aggregation Sensor

In deionized water, at concentrations below 1×10⁻⁶ M (in repeat units,RUs), PFPB emits predominantly in the 400 to 500 nm region, with afluorescence quantum yield (Φ) of 22%. Indeed, both the absorption(λ_(max)=380 nm) and the emission are nearly identical to that ofpoly(9,9-bis(6′-N,N,N,-trimethylammoniumbromide)hexyl)fluorene-co-alt-1,4-phenylene)(PFP), which lacks BT sites (see FIGS. 1A and 1B).¹⁸

Under dilute conditions, the emission of PFPB is dominated by the moreabundant oligo(fluorene-co-phenylene) segments. When [PFPB]>1×10⁻⁶ M,one observes the emergence of green emission (500 to 650 nm)characteristic of the BT sites. As [PFPB] increases, the green emissiongrows at the expense of the blue emission. These data indicateaggregation in the more concentrated regime, which leads to a reductionof the distance between polymer segments and enhances energy transfer tounits containing lower energy BT chromophores.^(19,20)

Example 3 General Procedure for FRET Experiments

The oligonucleotide used in the single stranded DNA study was 5′-TR-ATCTTG ACT ATG TGG GTG. The extent of hybridization was verified byvariable temperature absorbance spectroscopy. Fluorescence intensitieswere determined from the integrated areas under emission spectra of boththe donor and the acceptor (Texas Red). Measurements were carried out inwater at a fixed ss-DNA-TR concentration ([ss-DNATR]=2.0 E-8 M), byvarying the polymer with concentration varying from 0 M to 2.3 E-7 M.The excitation wavelength for PFB and PFPB was chosen at 380 nm, and theemission intensity was corrected to reflect the difference in opticaldensity for polymers. The PNA used in the DNA/PNA-Cy5 study correspondsto the sequence 5′-Cy5-CAG TCC AGT GAT ACG-3′. It was annealed at 2° C.below its melting temperature for 25 minutes in the presence of anequimolar amount of its complementary 15 base pair ss-DNAc (5′-CGT ATCACT GGA CTG-3′) and in an identical fashion with a noncomplementary 15base ss-DNAn (5′-ACT GAC GAT AGA CTG-3′). The absorbance of thehybridized strands was measured to determine concentration before usingin FRET experiments. The extent of hybridization was checked by variabletemperature absorbance spectroscopy.

Example 4 Detection of Aggregate Formation Using FRET

FIG. 1A shows the emission from PFPB/ss-DNA solutions upon addition ofss-DNA ([RU]=5×10⁻⁷ M, ss-DNA=(5′-ATC TTG ACT ATG TGG GTG CT), [ss-DNA]varies from 0 M to 2.7×10⁻⁸ M). The complexation of PFPB with ss-DNA(5′-ATC TTG ACT ATG TGG GTG CT) in water²¹ led to contraction andaggregation of polymer chains, a concomitant reduction of intersegmentdistances, and an increase in FRET to the BT sites. The isosbestic pointat 492 nm highlights the transition from blue to green emission withincreasing [ssDNA].

Example 5 FRET Using an Aggregation Sensor in a Multicolor Assay

FIG. 1B shows the emission spectra of PFP, and the absorption andemission of ss-DNA-TR (TR=Texas Red dye and ssDNA-TR=5′-TR-ATC TTG ACTATG TGG GTG CT). Note that the spectral overlap between the absorptionof TR and the green emission band of PFPB/ss-DNA is substantially largerthan with the emission of PFP. Therefore, we anticipated a larger valuefor the overlap integral in the Förster equation and more efficient FRETwith PFPB.²² Indeed, as shown in FIG. 2, the TR emission intensity as afunction of polymer concentration is much greater when PFPB is excited,relative to PFB (the value of Φ for TR is the same in the two sets ofsolutions). The spectra in FIG. 2 were measured by excitation at 380 nm,which selectively creates polymer-based excited states.

Based on the mechanistic information above, we postulated that PFPBcould be used in a three color DNA assay by using a PNA-C* strand (whereC* is a suitable fluorophore). PNA serves to provide a base sequencethat searches for a complementary ssDNA. However, because PNA isneutral, it is possible to use water without buffers or other ions thatare required to screen the negative charges during duplex formation.²³Since PNA-TR is not commercially available, we used PNA-Cy5(5′-Cy5-CAGTCCAGTGATACG) as the PNA-probe instead. The absorption andemission of Cy5 (λ_(abs)=648 nm, λ_(em)=681 nm) are similar to those ofTR. Hybridization of PNA-Cy5 with a complementary ss-DNA(ss-DNAc=5′-CGTATCACTGGACTG) endows the ss-DNAc/PNA-Cy5 duplex withmultiple negative charges. Complexation of ss-DNAc/PNA-Cy5 byelectrostatic forces to the positively charged PFPB allows for FRET fromthe polymer to Cy5 and should lead to red emission. In the case of anon-complementary ss-DNA (ss-DNAn=5′-ACTGACGATAGACTG), electrostaticcomplexation occurs only between PFPB and the ss-DNAn, which should giverise to emission from the BT units.

FIG. 3 shows the different emission colors observed in this detectionscheme. In water (pH=7.0), a solution of PFPB ([RU]=1.6×10⁻⁷ M) andPNA-Cy5 emits blue, indicating that no significant PFPB/PNA-Cy5complexation takes place. For the non-complementary situation, i.e.ss-DNAn and PNA-Cy5 (annealing protocols are done independently), greenemission is predominantly observed. Under similar conditions, whenss-DNAc/PNA-Cy5 is used, only red emission from the Cy5 units takesplace. These data indicate that FRET from PFPB to the Cy5 signalingchromophore is essentially complete.

The above examples report design guidelines for water soluble conjugatedpolymer structures that change emission color depending on their stateof aggregation. Complexation with oppositely charged polyelectrolytes(such as DNA) brings together polymer segments and encourages energymigration to low energy emissive sites (BT in the case of PFPB). Using aPNA-Cy5 probe strand, one obtains three different colors, depending onthe solution content: blue, in the absence of DNA; green, whennon-complementary ssDNA is present; and red, when the complementaryssDNA is found.

Example 6 Optimization of Polymer Structure and Quantitation ofPolynucleotides

It occurred to us that the shift in emission color of PFPB materialscould be used to determine the concentration of an aggregant such asdsDNA or ssDNA. In the absence of DNA, and under dilute conditions, thechains are isolated and emit blue. DNA-induced aggregation provides themolecular basis to modify FRET conditions and increase the greenemission, at the expense of the blue color. By measuring the increase ingreen emission and/or the decrease in blue emission and applyingcalibration curves one can therefore calculate the total DNAconcentration in the sample.

Initial efforts were directed towards optimization of polymer structure.PFPB structures with varying amounts of BT units were prepared as shownin Scheme 2. Suzuki cross-coupling mediated copolymerization of2,7-dibromo-9,9-bis(6′-bromohexyl)fluorene,1,4-phenylenebis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane), and4,7-dibromo-2,1,3-benzothiadiazole provides neutral precursor polymersin which the BT content is regulated by the ratio of2,7-dibromo-9,9-bis(6′-bromohexyl)fluorene and4,7-dibromo-2,1,3-benzothiadiazole. In a second step, quaternizationwith trimethylamine provides the water-soluble materials in greater than90% yields after isolation. Using the procedure in Scheme 2, PFPBpolymers were prepared with 1, 2.5, 5, and 7% BT contents. Efforts toobtain a material with 9% BT in this copolymer embodiment wereunsuccessful, because low solubility in water prevented completeconversion at the quaternization step. The polymers were pale yellow incolor and had virtually identical absorption spectra, with the maximaranging from 378 nm to 383 nm. Each polymer had a small shoulder at 440nm to 460 nm, attributable to the BT unit, which intensified withincreased BT incorporation.

FIG. 6 shows the changes in the photoluminescence (PL) spectra of PFPB₇(1.0×10⁻⁶ M in fluorene-phenylene repeat units, RUs) in water uponaddition of dsDNA in 6.2×10⁻⁹ M increments up to a concentration of9.6×10⁻⁸ M (DNA concentration is made relative to base pairs for dsDNAand bases for ssDNA). As [dsDNA] increases, there is a progressiveincrease of the emission band at 550 nm and a concomitant decrease ofthe band at 410 nm. An isosbestic point is observed at 490 nm. Thisshift in emission color is attributed to the energy transfer from themain component of the conjugated backbone to the BT sites uponinterpolyelectrolyte complex formation.

To obtain quantitative information on how the shift in emission colorcorrelates to [DNA], we define the parameter δ, as shown in Equation 1,which incorporates information on spectral characteristics and providesa means to normalize data across different instruments and opticalcollection conditions:

$\begin{matrix}{\delta = \frac{G_{n} - {G_{0}\left( \frac{B_{n}}{B_{0}} \right)}}{N}} & (1)\end{matrix}$

In Equation 1, G₀ is the integrated green fluorescence (500 nm to 700nm) in the absence of DNA, and corresponds to the emission tail ofPFPB_(x) in this range, G_(n) is the integrated green fluorescence atthe nth addition of DNA, B₀ is the integrated blue fluorescence (390 nmto 480 nm) in the absence of DNA, B_(n) is the integrated bluefluorescence at the nth addition of DNA and N is the normalizationfactor, obtained in our case by the emission (390 nm to 480 nm) of a1.0×10⁻⁶ M PFPB₇ which serves to compare measurements from instrumentsof different sensitivities and configurations.

A comparison of the responses of the different polymer compositions isgiven in FIG. 7( a) (left), where δ is plotted against [dsDNA]. Aboveall, for all structural compositions, there is a remarkable linearrelationship between δ and [dsDNA] in this concentration range.Additionally, there is a direct correlation between the BT content inthe backbone and the sensitivity of δ to [dsDNA]. As evidenced by thedifference in slopes, PFPB₁ is the least responsive and PFPB₇ is themost responsive, with lower detection limits of 6.0×10⁻¹⁰ M by (FIG. 7(b); right). That is to say, that at a given DNA concentration the shiftfrom blue to green is more pronounced in the seriesPFPB₇>PFPB₅>PFPB_(2.5)>PFPB₁. Such a trend is expected on the basis of agreater concentration of green emitters and the greater propensity foraggregation based on the lower solubility of polymers with a largerfraction of BT units. Further experiments therefore used PFPB₇.

Two significant features are illustrated in FIG. 8, which shows the 8response to 30 by dsDNA at three different PFPB₇ concentrations (in RUs:of 2.5×10⁻⁷ M, 5.0×10⁻⁷ M, and 1.0×10⁻⁶ M). First, the similar slopes inthe low concentration regime ([dsDNA]<5×10⁻⁸ M) reveal that the responseof the assay is independent of [PFPB₇]. Second, a leveling off of 8 vs.[dsDNA] takes place at higher [dsDNA]. This deviation from the initiallinear relationship suggests a stoichiometric limit for thepolyelectrolyte complex. Indeed, response saturation occurs when theRU/bp ratio is approximately 6, regardless of the total [RU]. Once thisratio is reached, there is no longer an increase in response from PFPB₇,and an upper limit for the response range is reached. We also note thatit is not feasible to use more concentrated PFPB₇ solutions to extendthe assay to more concentrated DNA regimes because PFPB₇ aggregationleads to BT emission in the absence of DNA.

Possible changes brought about by differences in DNA chain length wereexamined by comparing ssDNA and dsDNA ranging from 20 to 100 bases orbase pairs, respectively. FIG. 9 shows 8 as a function of [ssDNA] or[dsDNA] using PFPB₇ at each of the different DNA lengths. Significantly,the specific length of the DNA is not a factor in modifying the 8response, as each of the data points falls on the same line, regardlessof strand length. The responses of PFPB₇ for each DNA length are inclose agreement with each other, with a standard deviation of ˜8%. Asexpected, the slope for dsDNA is approximately twice that of the ssDNAdue to the doubling of charge density.

Example 7 Comparison to Commercial Dyes

PicoGreen and OliGreen are widely used commercially available probes forthe determination of [dsDNA] and [ssDNA]. Both reagents are based oncyanine dyes and are suspected to intercalate or otherwise bindirreversibly to DNA. This poses potential health and environmentalhazards; consequently, containment is important and disposal involvesthe adsorption of the dyes on activated charcoal, followed byincineration. Picogreen has a lower detection limit of 25 pg/mL(3.8×10⁻¹¹ M bp) of dsDNA and OliGreen has a lower detection limit of100 pg/mL (3.2×10⁻¹⁰ M b) of ssDNA. In comparison, PFPB₇ has a lowerdetection limit of 6.0×10⁻¹⁰ M by for dsDNA (FIG. 7( b), but exhibitsviability due to its excellent accuracy and repeatability. Additionally,PFPB₇ shows greater efficiency in terms of photoluminescence yield. Fora concentration of 2.0×10⁻⁸ M 30 by dsDNA, the relativephotoluminescence yield (emission intensity/photons absorbed) of PFPB₇is ˜3 times more efficient than PicoGreen and similar to that ofOliGreen. Another distinguishing feature of PFPB₇ is that it can be usedto quantify both ssDNA and dsDNA.

While PFPB₇ shows several potential advantages over PicoGreen andOliGreen, the basis of this quantification assay on attractiveelectrostatic interactions makes it more susceptible both to chargescreening and pre-aggregation in a buffered environment. Assays carriedout with salt concentrations of up to 1 mM are unaffected. However,higher salt concentrations give rise to pre-aggregation, resulting in ashift of blue to green emission prior to DNA addition.

In summary, changes in the PL color of an aggregation sensor can be usedto determine the concentration of ssDNA and dsDNA accurately, reliablyand efficiently. Highest sensitivity was observed with PFPB₇, whichcontains the highest concentration of BT units in the backbone whilekeeping the polymer sufficiently soluble in water. Use of PFPB₇aggregation induced by polyelectrolyte complexation eliminates thesafety hazards associated with intercalating dyes. Because the intensityof the signaling event is based on the charge density of a target,future efforts can be directed towards development of similarquantification schemes for other negatively charged polyelectrolytessuch as RNA, proteins or enzymes. Furthermore, we note that the range ofthis quantification approach can be broadened by the design ofnegatively charged PFPB analogs, such that biomolecules with multiplepositive charges can also be included as targets.

Methods for Examples 6 and 7

General details. ¹H and ¹³C NMR spectra were collected on a Varian Inova400 MHz NMR. Fluorescence measurements were recorded on a PTIQuantamaster 4 spectrometer or a Jasco FP-6300 spectrometer at 90°detection angles. Absorption measurements to quantify [DNA] and [RU]were recorded on a Beckman Coulter DU 800 UV-Vis spectrometer and aShimadzu UV-2401 UV-Vis spectrometer, respectively. DNA was purchasedfrom GenScript or Integrated DNA Technologies and HPLC purified.Sequences used in this study were a statistical distribution of the fourbases. PicoGreen and OliGreen kits were purchased from Molecular Probes.All FRET experiments were conducted in Milli-Pore water filtered with aBarnstead NANOpure II filtration system.

The percentages of BT groups incorporated in PFPB_(x) were estimated byabsorption spectra. Increased absorption of the BT group of the polymercould be seen as the BT to fluorene ratio in the polymer synthesis wasvaried. Quantum yield of fluorescence, (Φ_(PL), was measured usingfluorescein in water (pH 10) as the standard. Error associated with thismeasurement is ±0.05.

Synthetic details. Detailed procedures for the synthesis of PFPB₅ andprecursors have been previously reported. Syntheses of PFPB₁,PFPB_(2.5), PFPB₅, and PFPB₇ followed similar procedures, andcomposition was adjusted by varying the BT to fluorene feed ratio. TheNMR data is shown for PFPB₇ and its precursor.

PFPB₇ precursor. ¹H NMR (400 MHz, CDCl₃): δ=7.9-7.6 (m, 10.5H), 3.316(t, J=6.8 Hz, 4H), 2.112 (br s, 4H), 1.708 (m, 4H), 1.265 (br s, 4H),1.161 (br s, 4H), 0.807 (br s, 4H). ¹³C NMR (100 MHz, CDCl₃): δ=151.624,140.601, 140.373, 139.895, 127.817, 126.338, 121.543, 120.451, 55.419,42.961, 34.267, 32.856, 29.321, 27.993, 23.904.

PFPB₇. ¹H NMR (400 MHz, CDCl₃): δ=8.0-7.6 (m, 10.5H), 3.081 (m, 4H),2.902 (m, 18H), 2.131 (br s, 4H), 1.471 (br s, 4H), 1.103 (br s, 8H),0.645 (br s, 4H). Φ_(PL)=0.13.

General procedure for FRET experiments and analysis. Spectra wererecorded in the following manner: 1.5 mL of a solution of known [RU] wasprepared in a plastic cuvette. Before the introduction of DNA, thefluorescence was measured (390 nm to 700 nm), after which aliquots of aknown concentration of DNA were added. Spectra were recorded after eachsubsequent addition. Analysis of data followed Equation 1.

Although the invention has been described in some detail with referenceto the preferred embodiments, those of skill in the art will realize, inlight of the teachings herein, that certain changes and modificationscan be made without departing from the spirit and scope of theinvention. Accordingly, the invention is limited only by the claims.

REFERENCES

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1. A method of assaying a sample for an aggregant, the methodcomprising: providing an aggregation sensor soluble in a polar medium,the aggregation sensor comprising (a) a polymer comprising a pluralityof first optically active units forming a conjugated system, having afirst absorption wavelength at which the first optically active unitsabsorb light to form an excited state that can emit light of a firstemission wavelength, and a plurality of solubilizing functionalities;and (b) one or more second optically active units that can receiveenergy from the excited state of the first optically active unit; saidaggregation sensor comprising at least three first optically activeunits per second optically active unit, wherein a solution of theaggregation sensor emits detectably less light at the first emissionwavelength when the concentration of first optically active units withinenergy-transferring distance of at least one second optically activeunit is increased; combining the aggregation sensor and the sample insolution, wherein the aggregation sensor is present at a concentrationat which there is detectable emission at the first emission wavelengthin the absence of sample; contacting the aggregation sensor with lightof the first absorption wavelength; and detecting whether the opticalproperties of the aggregation sensor are altered in the presence of thesample.
 2. The method of claim 1, where detecting whether the opticalproperties of the aggregation sensor are altered in the presence of thesample comprises detecting if the aggregation sensor emits less light ofthe first emission wavelength.
 3. The method of claim 1, where thesecond optically active unit forms an excited state upon receivingenergy from the first optically active unit and emits light of a secondemission wavelength, and where detecting whether the optical propertiesof the aggregation sensor are altered in the presence of the samplecomprises detecting if the solution emits light of the second emissionwavelength.
 4. The method of claim 3, further comprising combining aprobe with the aggregation sensor and the sample in solution, whereinthe probe binds specifically to a particular species of a class ofaggregants that binds to the aggregation sensor, said probe attached toa fluorophore that receives energy from the second optically active unitwhen in proximity thereto and then emits light of a third emissionwavelength, where detecting whether the optical properties of theaggregation sensor are altered in the presence of the sample comprisesdetecting if the solution emits light of the third emission wavelength.5. The method of claim 4, wherein the amount of light emitted from thefluorophore is greater than can be achieved by direct excitation of thefluorophore with light of an appropriate wavelength.
 6. The method ofclaim 4, wherein the probe comprises a component selected from the groupconsisting of a polypeptide, a polynucleotide, and a peptide nucleicacid (PNA).
 7. The method of claim 1, further comprising combining anintercalating dye with the aggregation sensor and the sample in solutionunder conditions in which the dye intercalates with a sequence-specificduplex aggregate comprising a probe comprising a polynucleotide bound toa polynucleotide aggregant, wherein the intercalated dye exchangesenergy with at least one other optically active species present in theaggregate when formed.
 8. The method of claim 1, wherein the conjugatedpolymer comprises at least one optionally substituted repeat unitselected from the group consisting of 2,1,3-benzothiadiazole,benzoselenadiazole, naphthoselenadiazole,4,7-di(thien-2-yl)-2,1,3-benzothiadiazole, an olefin, acyano-substituted olefin, 2,7-carbazolene-vinylene, 2,7-fluorene, and4,4′-biphenyl.
 9. The method of claim 1, wherein the aggregation sensorcomprises a conjugated polymer comprising optionally substitutedpoly(fluorene-co-phenylene) repeat units with 2,1,3-benzothiadiazole asthe second optically active unit.
 10. The method of claim 1, wherein theaggregation sensor comprises a ratio of first optically active units persecond optically active units selected from the group consisting of atleast four first optically active units per second optically activeunit, at least six first optically active units per second opticallyactive unit, at least nine first optically active units per secondoptically active unit, and at least nineteen first optically activeunits per second optically active unit.
 11. The method of claim 1,wherein the aggregation sensor contains one second optically activeunit.
 13. The method of claim 1, wherein the first absorption wavelengthis about 488 nm.
 14. The method of claim 1, wherein the class ofaggregants is selected from the group consisting of polypeptides andpolynucleotides.
 15. The method of claim 14, wherein the aggregantdetected is a polynucleotide produced via an amplification reaction. 16.The method of claim 1, wherein the second optically active unit isgrafted to the conjugated system.
 17. The method of claim 1, wherein thesecond optically active unit forms part of the conjugated system. 18.The method of claim 1, wherein a single nucleotide polymorphism (SNP) isdetected in the aggregant.
 19. The method of claim 1, wherein expressionof a gene is detected upon detection of the aggregant.
 20. The method ofclaim 1, wherein a result is used to diagnose a disease state of apatient.
 21. The method of claim 20, wherein the use of the result todiagnose a disease state comprises reviewing or analyzing data relatingto the presence of an aggregant in a sample; and providing a conclusionto a patient, a health care provider or a health care manager, theconclusion being based on the review or analysis of data regarding adisease diagnosis.
 22. The method of claim 21, wherein providing aconclusion comprises transmission of the data over a network.
 23. Themethod of claim 1, where an aggregant is present in the sample and anaggregate is formed comprising the aggregant and the aggregation sensor.24. The aggregate formed by the method of claim
 23. 25. An aggregationsensor soluble in a polar medium comprising: (a) a polymer comprising aplurality of first optically active units forming a conjugated system,having a first absorption wavelength at which the first optically activeunits absorb light to form an excited state that can emit light of afirst emission wavelength, and a plurality of solubilizingfunctionalities; and (b) one or more second optically active units thatcan receive energy from the excited state of the first optically activeunit; said aggregation sensor comprising at least three first opticallyactive units per second optically active unit; wherein a solution of theaggregation sensor emits detectably less light at the first emissionwavelength when the amount of first optically active units withinenergy-transferring distance of at least one second optically activeunit is increased.
 26. A solution comprising the aggregation sensor ofclaim 25 and a solvent.
 27. A kit for assaying a sample for an aggregantcomprising: the aggregation sensor of claim 25; and a probe that canbind specifically to a particular species of a class of aggregants thatcan bind to the aggregation sensor, said probe attached to a fluorophorethat can receive energy from the second optically active unit and emitlight.